Injection Of Hematoporphyrin Derivative Research Articles

Establishing an experimental model of photodynamically induced anterior ischemic optic neuropathy

Numerous methods and drugs have been used to treat anterior ischemic optic neuropathy (AION); however, further investigations to determine the value of treatments for AION have been impeded by the lack of appropriate animal models of AION, significantly impacting on in-depth study of the disease. A rat model of AION was established, and corresponding functional changes of the fundus were observed using fundus fluorescein angiography (FFA), optical coherence tomography (OCT), and flash visual-evoked potential (F-VEP) in order to confirm the reliability of the AION model histopathologically. One day after model establishment, histopathology demonstrated that portions of the optic disc were highly edematous, with edema of nerve fibers and loose tissue, accompanied by displacement of the surrounding retina. At 23 days, the optic disc and surrounding nerve fiber layers had become thinner. None of the above-mentioned changes was observed in the laser, hematoporphyrin derivative (HPD), or naive groups. The results of fundus, FFA, F-VEP, and OCT-within 90 days after model establishment-confirmed that krypton red laser irradiation (647 nm), applied 2 h after HPD injection, can establish an ideal animal model of AION.

Clinical multi-colour fluorescence imaging of malignant tumours--initial experience.

The detection of malignant tumours relies on a variety of diagnostic procedures including X-ray images and, for hollow organs, endoscopy. The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents, such as haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation (transitional cell carcinoma of different stages including carcinoma in situ), ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). Finally, the extent of in situ and invasive carcinomas of the breast was investigated in surgically excised specimens from patients that received a low-dose injection of HPD 24 h prior to the study. The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s, was mixed with a normal colour video image of the tissue. A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in in vitro investigations of resected breast cancer. The initial clinical experience of using multi-colour fluorescence imaging has shown that the technique has the potential to reveal malignant tumour tissue, including non-invasive early carcinoma and also precancerous tissue. Further investigations are needed to fully develop the method.

Fluorescence localization in tumour and normal brain after intratumoral injection of haematoporphyrin derivative into rat brain tumour

In the intracerebral 9L rat gliosarcoma, the spatial distribution of the photosensitizer haematoporphyrin derivative (HpD) was studied after intratumoral injection. The fluorescence volume was measured in histological sections from 10 min up to 5 days after injection. Complete sensitization of the tumours could not be achieved by slow stereotactical injection of 4 mm 3 HpD (mean HpD fluorescence volume, 13 ± 11 mm 3). Larger parts of the tumour could be loaded with HpD (39 ± 23 mm 3, p = 0.0001) by increasing the injection velocity and the volume to 50 mm 3. Again, complete sensitization of the tumours was not achieved during a time scale of 5 days afte intratumoral injection. Although fluorescence volume did not change significantly with time, it was influenced by the injection site within the tumour. Injection of HpD within 1 nm from the tumour border resulted in significantly smaller fluorescence volumes in the tumour than injection into the tumour centre. Large injection volumes caused an increased leakage of HpD to normal brain, leading to the loss of selectivity of photosensitizer content and the occurrence of dark toxicity of normal brain while the tumours still appeared vital.

Fluorospectral study of the rat brain and glioma in vivo

An animal model of cerebral glioma was utilized by implanting C6 glioma cells into the brains of adult Wistar rats. Once tumors developed to 7-12 mm in diameter, we conducted continuous fluorimetry monitoring of glioma up to 24 hours using a fibre-optic system connected to an intensified multichannel photodetector after an intravenous injection of hematoporphyrin derivative (HPD) into the rats. The intensity of the fluorescence in normal brain reached a plateau 6 hours after intravenous injection of HPD while that in glioma reached a plateau 80 minutes after injection. These fluorescence intensities of glioma, brain adjacent to tumor (BAT), and surrounding normal brain were measured in vivo 24 hours after intravenous administration of 5 mg/kg of HPD. The ratio of fluorescence intensities between glioma and brain was 6.1 while the ratio between BAT and brain was 3.9. There were no obvious differences in shapes between the spectra of the natural fluorescence (autofluorescence) of rat glioma and brain but the intensity of autofluorescence was much weaker in glioma. There are many problems in spectroscopic studies of biological tissues in vivo. It cannot be overemphasized that very strict criteria must be applied in order to get accurate data. Fluorescence from HPD administration may be used to discriminate tumor tissue from surrounding normal brain tissue during operation if the measuring conditions could be kept constant. It is important to understand the photospectral properties of glioma and brain tissue in order to get the most benefits in clinical application of light-induced fluorescence or photoradiation therapy.

Photodynamic Therapy of Esophagogastric Tumor with Intra-Tumoral Injection of Hematoporphyrin Derivative

From January 1988 to June 1989, twelve patients with endoscopically proven esophagogastric tumor were accepted for photodynamic therapy (PDT) at Taipei Municipal Jen-Ai Hospital for a total of 26 sessions. To avoid undesirable systemic photosensitization side effects, an intra-tumorous dose of 10-20mg (approximately 5mg/cm^2) hematoporphyrin derivative (HpD) was given during the endoscopy. Two or three days after the HpD injection, the tumor areas were photoradiated with 630 nm light with a total energy density of 120 J/cm^2. The procedure was repeated at 7 day intervals until the tumor was no longer visible or was remarkably reducd in size. The overall response rate to this therapeutic effect was 83% with satisfactory effect at 58%. In this series, 5 cases were diagnosed in the early stages of cancer. After treatment, 3 cases responded completely and 2 cases partially. During the entire course, no systemic photosensitization or severe complications were observed. Our preliminary results suggest that local photosensitization by HpD could be used as a therapy modality for treatment of neoplastic esophagogastric lesions although it will have to be confirmed by a larger number of cases and follow up a longer period.

In vivo to post-mortem change in tissue penetration of red light

For precise light dosimetry in photodynamic therapy (PDT), the light penetration characteristics of the tissue concerned need to be assessed. Several attenuation coefficients have so far been measured using 633 nm wavelength red light and human tissue obtained from autopsy or surgery. However, the validity of such ex vivo derived values remains uncertain, and hence is evaluated further in this study. Using a rat thigh muscle model, the tissue transmittance of 633 nm red light was compared between in vivo and ex vivo. Transmittance (in vivo) following preliminary injection of 40 mg kg−1 haematoporphyrin derivative (HPD) was also measured in muscle and liver. The effective attenuation coefficient (⌆eff) ex vivo in muscle was 0.98±0.06 mm−1 and in vivo 0.97 ± 0.05 mm−1. Values agreed within experimental error indicating that in this tissue, changes from in vivo to post-mortem produced no alteration in optical penetration at 633 nm. Preliminary injection of HPD did not influence the penetration depth in muscle although significant changes were observed in an organ of high porphyrin avidity, the liver (0.68±0.08 mm vs 0.40±0.08 mm with HPD). Until techniques for in vivo measurements of light attenuation coefficient become available, the use of ex vivo derived values with 633 nm wavelength light would seem appropriate.

Photodynamic therapy is potentiated by Co60 and intratumoral injection of hematoporphyrin derivative

Hematoporphyrin derivative injected directly into a subcutaneous rat glioma resulted in a significant greater tumor growth inhibition than hematoporphyrin derivative injected parenterally upon stimulation by light, Co60 or by combination of the above. The photodynamic effect was analyzed in an in vivo and in an in vitro clonogenic assay. The directly injected hematoporphyrin derivative without external activation inhibited tumor growth to 34%. Light and directly injected HPD inhibited tumor growth to 3%, whereas 4, 8 and 16 Gy of Co60 produced a growth inhibition to 11%, 0.05% and 0.00% respectively. This cytotoxic effect of the ionizing radiation is further potentiated by the addition of light, resulting in a growth inhibition to 0.1%, 0.00% and 0.00% respectively for the three corresponding radiation doses. The direct intratumoral injection of HPD minimizes the side effects and increases the effect of the photodynamic therapy as compared to the parenterally administered HPD. This direct injection modality could be of potential value in the treatment of human gliomas or other tumors.

Photodynamic therapy of C3H tumours in mice: Effect of drug/light dose fractionation and misonidazole

C3H mammary carcinomas transplanted to the feet of mice were treated with haematoporphyrin derivative (HPD) or Photofrin II(PII) and laser light at 630 nm. While fluence rates lower than 100 mW cm−2 gave minimal hyperthermic effects, a slight but significant growth delay was observed in unsensitized tumours exposed to a fluence rate of 150 mW cm−2 which induced tumour temperatures in the range 40–50°C. Different modes of fractionation of the light fluence and of the HPD dose were tested but were found to give poorer rather than better results than the application of a single light exposure 24 h after intraperitoneal injection of HPD. Different PII doses were applied together with different light fluences, keeping the product of the drug dose and light fluence constant. In the dose range 6.25–50 mg/kg body weight the resulting effect on tumours was constant, allowing for a slight effect of hyperthermia at the highest light fluences, and possibly a photodegradation of PII. Misonidazole given before photodynamic treatment (PDT) slightly reduced the effect of PDT on the tumour growth. When given after PDT, however, misonidazole improved the therapeutic results significantly.

Therapeutic in vivo and in vitro effects of argon dye laser and hematoporphyrin derivative in lung cancer.

In clinical and experimental study the therapeutic efficacy of argon dye laser irradiation with hematoporphyrin derivative (HpD) was evaluated in lung cancer. A total of 14 lung cancer cases including 12 squamous cell, 1 adeno- and 1 small cell carcinomas were irradiated superficially 48 hours or more after i.v. injection of 3 mg/kg of HpD (100-300 mW, 20-30 min.). Human adenocarcinoma cells implanted subcutaneously into nude mice were photoirradiated (200 mW, 20 min.) 48 h after i.p. injection of HpD. The in vitro effect of phototherapy was studied in the same cell line after incubation in medium containing HpD compared to untreated, only irradiated or only in HpD incubated cells. Among 3 early stage squamous cell carcinoma cases 2 complete and 1 partial remissions were obtained. Among 11 cases including 10 with advanced and 1 with recurrent disease 7 demonstrated partial remission. In vivo, two of 9 mice had a complete tumor remission. In the in vitro study, tumor cells incubated in 30 micrograms HpD/ml showed severe cytotoxic effects resulting in cell death 12 hours after photo irradiation, whereas cells incubated in 30 micrograms HpD/ml only regenerated after initial cytotoxic reaction. Laser irradiation only had no effects. HpD phototherapy demonstrated a considerable antitumor efficacy and must be considered as one of the promising endoscopic treatments in cases with early stage primary lung cancer.

Morphological changes of tumor microvasculature following hematoporphyrin derivative sensitized photodynamic therapy.

Abstract There is increasing evidence that the tumor microvasculature is affected during porphyrin photodynamic therapy. In the following study, the effect of hematoporphyrin derivative photodynamic therapy on tumor microvasculature was studied by electron microscopy. Urothelial tumors, implanted subcutaneously in rats, were exposed to red light (> 590 nm; 360 J cm−2) 72 h after injection of hematoporphyrin derivative at a dose of 5 mg kg−1 of body weight. Prior to sampling, in vivo perfusion was carried out using a polyvalent cation, lanthanum, in 3% glutaraldehyde to define the endocapillary layer of endothelial cells. Samples of tumors were collected at 0, 1,2 and 4 h after completion of photodynamic therapy. Histological changes in endothelial cells were evident immediately following completion of light exposure. Immediate morphological changes included absence of the endocapillary layer as well as mitochondrial degeneration. The changes within tumor cells followed the changes within the microvasculature. This study indicates that the endothelial cell of tumor tissue is an important target of photodynamic therapy and may be responsible for the blood flow changes reported previously.

Photosensitizers in dermatology.

Systemic injection of hematoporphyrin derivative (HpD) in contribution with visible light (red or blue-green) delivered by laser was used to treat a patient with psoriasis. The psoriatic lesions responded vigorously to laser treatments, forming eschars by 1 week post irradiation. In contrast, only minimal erythema was observed in the noninvolved, clinically normal appearing skin. Two approaches for localized HpD administration were investigated in the guinea-pig and minipig models as a means of achieving local photodynamic effects. Intracutaneous injection of HpD produced localized cutaneous photosensitization with either UVA or red light. Azone increased percutaneous penetration of HpD in human skin in vitro. Topical application of HpD and irradiation with UVA produced localized cutaneous photosensitivity and inhibition of epidermal DNA synthesis.

In Vivo Mediator Release and Degranulation of Mast Cells in Hematoporphyrin Derivative-Induced Phototoxicity in Mice

This study was designed to assess the role of the mast cell in the early phase of hematoporphyrin derivative (HPD)-induced phototoxicity. BALB/c mice were rendered phototoxic by i.p. injection of hematoporphyrin derivative, followed by exposure to 13.6 kJ/m2 of 400-410 nm radiation. The phototoxic response was quantified by measurement of ear thickness immediately before the irradiation, and at 0, 0.5, 1, 1.5, and 2 h after. At these time-points, determinations of serum histamine and plasma leukotriene C4 levels and histologic examination of the ears were undertaken. Mice injected i.p. with buffered saline and subsequently irradiated served as controls. In mice exposed to HPD and radiation, a maximal peak increased ear-thickness of 125.7 +/- 14.4% (mean +/- SEM) was noted at 2 h; this was associated with a net increased serum histamine of over 120% and histologic evidence of mast cell degranulation. In addition, moderate increases in plasma levels of leukotriene C4 were observed at 0 h and 1.5 h in the HPD- and irradiation-treated animals. These data provide direct evidence for the participation of mast cells in the early phase of HPD-induced phototoxicity.

Photodynamic therapy for malignant glioma by argon laser. Basic study and clinical trials.

This study was designed to assess the therapeutic application of photodynamic or photoradiation therapy (PDT) using hematoporphyrin derivative (HPD) and argon laser for human glioma. Fluorescence assays of HPD were obtained using a microfluorescence photometer mounted on a microscope (Leitz, West Germany). An optimal time and exposure dose after injection of HPD were evaluated in the experimental brain tumors, 9L glioma cells implanted into the rat brain. An argon laser with 488 nm wavelength was used as a light source. The amount of HPD in the experimental brain tumor was large between 24 and 96 hours after injection of 10 mg/kg of HPD, and it was also confirmed in the human patients with glioma. The different experimental brain tumor treatment groups, using a dose of 383 joules/cm2. sec of HPD-PDT, were evaluated by mean life spans. They were 22 ± 6 days in a non-treated group, 27 ± 5 days in a group treated by operation or surgical removal alone, and 69 ± 5 days in a group given intraperitoneal and local administration of HPD and operation-PDT. An alteration of the tissue temperature during HPD-PDT exposure of 450 joules/cm2. sec was studied. Tissue temperature increased between 4°and 9°C within normal tissue 4 mm from the injured area. Sixteen patients with glioma were treated by HPD-PDT. All the patients underwent surgical removal of the tumor, followed by PDT 72 hours after intravenous injection of 5 mg/kg of HPD. The radiation exposure was between 243 and 1, 564 joules/cm2 ⋅sec. Six patients with malignant glioma revealed a recurrence of the tumor in the exposed area within 6 months after PDT. They received a low dose, less than 311 joules/cm2. sec, or had a large residual volume of the tumor after surgery. It is suggested that the argon laser exposure should be above 343 joules/cm2. sec in energy for a remaining tumor of small volume.

Photodynamic therapy for head and neck cancer xenografts in athymic mice.

This study examines efficacy and optimal treatment variables of photodynamic therapy (PDT) for human head and neck squamous cancer (HNSC) xenografts in athymic mice. Two and four days after injection of hematoporphyrin derivative (HPD), tumors were illuminated with red light from an argon-dye laser. Sixty-three tumors were treated. With HPD dose and light intensity constant at 7.5 mg/kg and 100 mW/cm2, respectively, the extent of tumor necrosis was strongly dependent on duration of light exposure. There was no substantial difference in results for 30- and 60-minute treatment durations between animals injected with HPD 2 and 4 days before treatment. After 30 minutes treatment time, responses were seen in 8 of 10 mice (2 days post-HPD) and 11 of 12 mice (4 days post-HPD). After 60 minutes treatment time, toxicity was high. We conclude that, in this model, PDT is effective in selective killing of HNSC. For future comparison studies in this model, if the indicated HPD dose and light intensity are used we recommend a 2-day delay after HPD injection and a light exposure duration of 30 minutes.

Experimental aspects of in vitro and in vivo photochemotherapy

The selectivity of in vitro photodynamic reactions and the in vivo effects induced by PRT, whether the irradiation is applied interstitially or externally, still remains unclear. In vitro studies were performed using leukemic cell lines and syngeneic normal hemopoietic progenitors. For these, cells incubated with hematoporphyrin derivative (HPD) and non-incubated cells were irradiated with an argon laser. Data were obtained as the count of cell colonies found after a 7-day incubation period on semi-solid collagen gel medium. In vivo studies employed the HT 29 tumor model grafted into nude mice. Both animals injected with HPD and non-infected controls were irradiated with a dye laser pumped by an argon laser (Coherent) using a 400 micron optic fiber located either at a distance of 65 mm from the skin or inserted into the tumor. The temperature increase occurring during PRT was measured using non-absorbing thermocouples. In vitro, after HPD treatment and argon irradiation leukemic cells showed a greater phototoxicity (greater than 2 log10) than did the normal cells (0.25 log10). In vivo, when the heat rise is very similar (less than 4 degrees C) in both the tissues irradiated externally and those irradiated interstitially after HPD injection, histological examination of these did not reveal any quantitative differences (90% of tumor mass). These results are discussed.

Tumor-localizing and photosensitizing properties of hematoporphyrin derivative in hamster buccal pouch carcinoma

The tumor-localizing and photochemotherapeutic properties of hematoporphyrin derivative (HPD) were examined in 7, 12 dimethylbenzanthracene (DMBA)-induced oral cancers in the Syrian hamster. Oral tumors in hamsters injected with HPD (50 μg per gram of body weight) exhibited bright salmon pink fluorescence when exposed to long-wave ultraviolet light 24 hours after intraperitoneal HPD injection. Adjacent tumor-free mucosa did not fluoresce. Similarly, tumors not treated with HPD, normal mucosa treated with HPD, and normal mucosa not treated with HPD did not fluoresce. Tumors in animals that received HPD and photochemotherapy (PCT) were examined for gross and microscopic pathologic changes following the phototreatment. Tumors displayed edema, hemorrhage, and cellular necrosis that progressed with the time of sampling after photochemotherapy. Complete tumor necrosis was evident in the majority of oral tumors 24 hours after HPD PCT.

脳腫瘍治療にArgon Laser (488nm) 照射によるHPD-PDT法を応用する場合の問題点

This study was designed to assess the therapeutic application of photodynamic therapy (PDT) and hematoporphyrin derivative (HPD) by argon laser with 488 nm wavelength for human glioma. The tissue exposed by argon laser revealed a heterogenously degenerative change of the tissue within the depth of 1mm to 7mm on the dose of less than 311 joules. Tissue temperature increased between 4 and 9°C within a 4mm distant normal tissue from an exposed area during HPD-Argon PDT. The HPD density was observed in the peripheral area of the experimental brain tumor by an intraarterial injection of Mannitol and HPD. The coagulation necrosis was observed within a range of 5.3 mm on diameter by interstitial irradiation.

Cerebral photosensitisation by haematoporphyrin derivative. Evidence for an endothelial site of action.

Exposure of the cranium to white light in mice that had been given haematoporphyrin derivative (HpD) led to a rapid onset of vasogenic cerebral oedema, cerebral necrosis, coma and death. Selectivity of the initial damage for endothelium was suggested by (a) early breakdown (less than 1 h) of the blood-brain barrier (BBB) as shown by increased permeability to Evans blue (b) separation and increased vesiculation of endothelial cells at 2 h and (c) endothelial cell pyknosis at 3--4 h in small vessels next to apparently undamaged neurones and neuroglia. There was no damage to myelin sheaths, and astrocytes showed only end-feet oedema, a reaction to exudation of protein-rich fluid. Within a few hours of illumination, most cells in the illuminated area were necrotic. Cerebral photosensitivity persisted for at least 12 weeks after a single injection of HpD. Our results suggest that the primary site of damage in the brain is the endothelium of small vessels, and that HpD remains associated with this for a remarkably long time. These findings are relevant to the mechanisms by which photodynamic therapy damages other tissues, including neoplasms, and particularly to the possible application of this treatment to brain tumours.

Uptake and retention of hematoporphyrin derivative in an in vivo/in vitro model of cerebral glioma.

Photoirradiation treatment depends on exposing tumors to a photosensitizer and light to achieve selective tumor kill. We evaluated the kinetics of uptake of a photosensitizer, hematoporphyrin derivative (HpD), in an animal model of cerebral glioma to ascertain the optimal time for photoirradiation therapy. Animal models of cerebral glioma were established by implanting cells from the rat C6 glioma cell line into rat brains or as xenografts in adult mouse brains. C6 cells (10(7] injected into the frontal lobe of adult Wistar rats produced intracranial tumors greater than 5 mm in diameter in 90% of the animals at 21 days. Tumors greater than 4 mm in diameter developed in adult mouse brains within 14 days after 10(6) cells were implanted into the frontal lobe. These two tumor models were used to investigate the localization of HpD. After HpD administration, its presence was detected in fresh, unfixed specimens by fluorescence emission after excitation with an ultraviolet lamp. Fluorescence was determined quantitatively by an image analysis method using an optical data digitometer. The fluorescence, which was highly localized selectively to the intracerebral tumor, was just detectable 5 minutes after an intravenous injection of HpD. Patchy, bright fluorescence was evident 4 hours after injection, and the tumor was uniformly fluorescent after 6 hours. A minimal dose of 0.5 mg of HpD per kg of body weight was necessary to produce detectable fluorescence, and the dose of HpD necessary to produce detectable fluorescence was 4 mg/kg of body weight. The intracarotid route of administration was unsatisfactory because seizures were induced, and intrathecal injection did not produce significant fluorescence in the tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Photosensitization of mitochondrial adenosine-triphosphatase and adenylate kinase by hematoporphyrin derivative in vitro.

Mitochondrial ATPase and adenylate kinase activity of hepatoma cells were inhibited by hematoporphyrin derivative (HPD) followed by photoirradiation. Inhibition of ATPase activity was a dose- and time-related event. Malonaldehyde (MDA) content of mitochondrial membranes was markedly increased by HPD plus light. The content of mouse liver microsomal cytochrome P-450 was greatly increased after intraperitoneal injection of HPD for 4 days (5 mg/kg/day). The liver weight, and levels of liver microsomal G-6-phosphatase, MDA and triglyceride (TG) showed no difference in treated vs. control animals. The data presented here demonstrate that mitochondria may be a sensitive site of action of HPD photosensitization, and inactivation of ATPase and adenylate kinase may be an important contributing factor to tumor cell damage and death.