Photodynamic therapy in the treatment of malignant tumours: an analysis of 540 cases

Malignant tumours (540 cases), including tumours of the lung, oesophagus, cardia, stomach, rectum, bladder, other urinary genital organs, face and mouth, eyes, ear, nose and throat (ENT), head and neck, breast and skin, were treated using photodynamic therapy (PDT) between 1982 and 1985 in Beijing. All of the cases were identified pathologically and the patients received haematoporphyrin derivative (HPD) (5 mg kg-1) intravenously 48-72 h prior to PDT. An argon-pumped dye laser emitting at 630 nm was used for the treatment. The results were as follows: complete response (CR) was obtained in 227 cases (42.1%), partial response (PR) was obtained in 114 cases (21.1%), mild response (MR) was obtained in 120 cases (22.2%) and 79 cases (14.6%) showed no response (NR). The effectiveness of PDT in the different organs was compared. HPD fluorescence was examined in 409 cases of malignant tumours: 344 lesions (84.1%) revealed red fluorescence (positive reaction), 32 gave an equivocal response and 33 gave a negative reaction. Positive fluorescence was seen in all types of malignant tumour in our study. Indications and limitations of PDT for the different organs are discussed and compared.     

Photodynamic therapy in the treatment of actinic keratosis

The efficacy of photodynamic therapy (PDT) with 5-aminolevulinate and methyl aminolevulinate in the treatment of actinic keratosis has been demonstrated in a large number of clinical studies over the last several years. Here, we recapitulate the major findings, comparing the various photosensitizers, light sources and therapeutic regimens, and present a retrospective analysis of 142 own cases treated with 259 PDTs. In addition, we also discuss the value of PDT in comparison with cryotherapy or 5-fluorouracil. The efficacy and the low risk of side effects of PDT have resulted in a high patient preference in clinical trials.     

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid–base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid–base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.     

Photodynamic therapy treatment of early oral and laryngeal cancers

Photodynamic therapy (PDT) is a nonsurgical, minimally invasive treatment that uses a light source to activate light-sensitive drugs or photosensitizers in the treatment of cancer and other diseases. PDT has been successfully employed to treat early carcinomas of the oral cavity and larynx preserving normal tissue and vital functions of speech and swallowing. Two hundred seventy-six patients with early carcinomas of the oral cavity and larynx were treated from 1990 to 2006. Cure rates with a single treatment for early laryngeal and oral cancers were 91% and 94%, respectively. PDT is an effective primary and alternative treatment modality for early oral cavity and laryngeal cancers.     

Photodynamic therapy with topical delta-aminolaevulinic acid for the treatment of plantar warts

Treatments currently employed for plantar warts are often painful (electrosurgery, cryotherapy) and not always effective (keratolytic agents). In this paper we investigate the effect of photodynamic therapy (PDT) with topical delta-aminolaevulinic acid (ALA) on plantar warts. In order to remove the superficial hyperkeratotic layer of the warts an ointment containing 10% urea and 10% salicylic acid was applied for 7 days. After gentle curettage, a cream containing 20% ALA was applied under an occlusive dressing for 5 h on 64 warts, while 57 warts (controls) received only the vehicle. Both the ALA-treated warts and the controls were irradiated using a visible light lamp (with a range of 400-700 nm, peaking at 630 nm). The light dose was 50 J/cm(2). Patients were followed-up for 22 months. Two months after the last irradiation session 48 (75.0%) out of 64 ALA-PDT treated warts had resolved. By contrast only 13 (22.8%) of the 57 control warts had done so. During the treatment a few patients complained of a mild burning sensation. The absorption of ALA by the verrucous tissue was demonstrated by in vivo fluorescence spectroscopy. This study shows that topical ALA-PDT can be an alternative treatment for plantar warts. Further studies will be necessary in order to optimize the concentration of ALA and duration of treatment.     

Photodynamic therapy for periodontal diseases: state of the art

BACKGROUND: Photodynamic killing of periodontopathogenic bacteria may be an alternative to the systemic application of antibacterial drugs used in the treatment of periodontal diseases. Even though the method is still in the experimental stage, increasing bacterial resistance problems may promote the introduction of photodynamic therapy (PDT) into periodontal practice. AIM: In this review a literature survey is given of PDT as seen from a periodontal perspective. METHODS: In this review, the present knowledge and experience of PDT is summarized. Literature data are presented on drawbacks of conventional antibiotics, the mechanism of PDT, bactericidal effects of PDT as well as results of clinical efforts. The future prospects of the method are discussed. RESULTS: The application of photosensitizing dyes and their excitation by visible light enables effective killing of periodontopathogens. Encouraging studies using PDT in periodontitis and in peri-implantitis are known. CONCLUSION: Even though PDT is still in experimental stages of development and testing, the method may be an adjunct to conventional antibacterial measures in periodontology. Clinical follow-up studies are needed to confirm the efficacy of the procedure.     

Monitoring of impending myocardial damage after pleuropneumonectomy and intraoperative photodynamic therapy for malignant pleural mesothelioma using biochemical markers

In five patients who were treated for malignant pleural mesothelioma (MPM) with pleuropneumonectomy and intraoperative photodynamic therapy (IPDT), impending myocardial damage was monitored using ECG, the classical biochemical markers (creatine kinase [CK], total activity; CKMB, mass; and myoglobin), and the new cardiac markers troponin I (cTnI) and troponin T (cTnT). In the peroperative and postoperative period all classical markers were elevated, in contrast to cTnI and cTnT, because of the concomitant skeletal muscle damage. Sequential electrocardiogram monitoring showed no signs of myocardial damage. From this study in patients with MPM treated with pleuropneumonectomy and IPDT it can be concluded that measurement of cTnI and cTnT for the detection of myocardial damage is more suitable than measurement of the classical markers.     

Photodynamic therapy: A special emphasis on nanocarrier-mediated delivery of photosensitizers in antimicrobial therapy

Resistance to antimicrobial drugs is an impending healthcare problem of growing significance. In the post-antibiotic era, there is a huge push to develop new tools for effectively treating bacterial infections. Photodynamic therapy involves the use of a photosensitizer that is activated by the use of light of an appropriate wavelength in the presence of oxygen. This results in the generation of singlet oxygen molecules that can kill the target cells, including cancerous cells and microbial cells. Photodynamic therapy is shown to be effective against parasites, viruses, algae, and bacteria. To achieve high antimicrobial activity, a sufficient concentration of photosensitizer should enter the microbial cells. Generally, photosensitizers tend to aggregate in aqueous environments resulting in the weakening of photochemical activity and lowering their uptake into cells. Nanocarrier systems are shown to be efficient in targeting photosensitizers into microbial cells and improve their therapeutic efficiency by enhancing the internalization of photosensitizers into microbial cells. This review aims to highlight the basic principles of photodynamic therapy with a special emphasis on the use of nanosystems in delivering photosensitizers for improving antimicrobial photodynamic therapy.     

Photodynamic therapy of cancer: Second and third generations of photosensitizers

Photodynamic therapy of cancer (PDT) at early stages of various types of cancer is based on the use of the phototoxicity of photosensitizers (PS) which appears upon their excitation by light in tumor tissues. The review concentrates mainly on the data for the second and third generations of photosensitizers: the structure of PS, their photophysical and photochemical properties, and some results of theirin vitro andin vivo application. A carefully designed PS should exhibit the following properties: long-wave absorption, good singlet oxygen quantum yield, an intramolecular polarity axis, and lowin vivo (photooxidation) stability. Uncharged PS that are lipophilic, positively charged, and capable of binding with monoclonal antibodies are discussed as an example.     

Photodynamic therapy: regulation of porphyrin synthesis and hydrolysis from ALA esters

Photodynamic therapy (PDT) is a tool for the treatment of certain cancerous and pre-cancerous conditions. The natural precursor of porphyrins 5-aminolevulinic acid (ALA) has been extensively used as a pro-photosensitiser in PDT. ALA's poor permeability has been enhanced by chemical esterification with aliphatic alcohols. Some of the ALA esters proved to be more efficient than ALA for porphyrin synthesis. In the present work we studied the nature of porphyrin synthesis regulation from the ALA esters Hexyl-ALA (He-ALA) and R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) in an adenocarcinoma cell line. We found that He-ALA is incorporated into the cells at a higher rate, followed by THP-ALA and ALA, whereas ALA and ALA esters efflux at the same rate mediated by passive diffusion. Although ALA entrance to the cell might be regulatory at low concentrations, ALA derivative uptake is not a limiting factor. At high concentrations, the regulation of ALA conversion into porphyrins is driven by the enzyme porphobilinogenase, whereas ALA esters hydrolysis is regulated by esterases. The key conclusion of this contribution is that the use of ALA esters has to be limited to low concentrations where no regulation on porphyrin synthesis takes place.     

Photodynamic therapy induces selective extravasation of macromolecules: Insights using intravital microscopy

Photodynamic therapy (PDT) with Visudyne^® acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature.Fluorescein isothiocyanate dextran (FITC-D, 2000 kDa), a macromolecular agent, was intravenously injected 10 min before (LK0 group, n = 14) or 2 h (LK2 group, n = 16) after Visudyne^®-mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n = 8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500 s after injection. We also monitored PDT-induced leukocyte rolling in vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers.In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT-treated areas as compared to the surrounding non-treated areas (p < 0.0001). There was no FITC-D leakage in the control animals. Animals from the LK0 group had significantly less FITC-D extravasation than those from the LK2 group (p = 0.0002). In the LK0 group FITC-D leakage correlated significantly with the inflammation (p < 0.001).At the selected conditions, Visudyne^®-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo.     

Validation of INAA and AAS analysis protocols for whole blood: A study on cardiovascular and malignant hypertensive patients

Instrumental neutron activation analysis (INAA) and atomic absorption spectrophotometry (AAS) were used to verify the protocols for the quantification of zinc and magnesium in human whole blood. A study was conducted on group of volunteers consisting of 131 patients having cardiovascular diseases (CVD) and 23 malignant hypertension (MH) patients along with 432 control subjects with 218 male and 214 females. The elemental data of these elements has been exploited to establish the base line values in control subjects. The possibility of any relationship between blood Mg and Zn levels with CVD and MH has also been investigated. The mean blood Mg and Zn levels were found to be depleted in both CVD and MH patients as compared to normal subjects. In our findings Mg and Zn were found to have an inverse relation with systolic and diastolic pressure. The reliability of the methods was checked by the concurrent analysis of the IAEA reference material (RM) employing the optimized INAA and AAS protocols. The determined values by both techniques were found to be in good agreement with the IAEA reference values. The elemental data in whole blood samples of normal volunteers has also been compared with Mg and Zn contents reported by other countries.     

Application of accelerator mass spectrometry to macromolecules: preclinical pharmacokinetic studies on a polybisphosphonate

Data on the use of accelerator mass spectrometry (AMS) in conjunction with in vivo studies of macromolecular drugs are scarce. The present study shows the versatility of this technique when investigating the pharmacokinetics (PK) of a macromolecular drug candidate, a polybisphosphonate conjugate (ODX). The aforementioned is a polymer (molecular weight ~30 kDa) constituting a carbohydrate backbone with covalently linked ligands (aldendronate and aminoguanidine) and is intended for treatment of osteoporosis and the therapy of bone metastasis from prostate cancer. The conjugate is prepared through partial oxidation of the carbohydrate and sequential coupling of the ligands by reductive amination. ¹⁴ C was incorporated in the conjugate by means of coupling a commercially available ¹⁴ C‐lysine in the conjugation sequence. Fifteen rats were injected intravenously with ¹⁴ C‐labelled ODX (150 µg, 14 Bq/rat) and blood samples were collected at 1, 2, 4, 6, and 24 h post‐injection (3 rats/time point). Liver, spleen and kidney samples were collected at 4 and 24 h post‐injection. Blood from each time point (triplicate) were collected for AMS measurement determining the isotopic ratio (¹⁴ C/¹² C) and consequently the drug concentration in blood. ODX showed a transient presence in blood circulation; 93% of the total dose was cleared from the circulation within 1 h. The half‐life after 1 h was estimated to be about 3 h; 0.7% of the administered ¹⁴ C dose of ODX remained in circulation after 24 h. The major ¹⁴ C accumulation was in the liver, the spleen and the kidneys indicating the probable route of metabolism and excretion. This study demonstrates the versatility of AMS for pharmacological in vivo studies of macromolecules. Labelling with ¹⁴ C is relatively simple, inexpensive and the method requires minimal radioactivity, eliminating the need for radioprotection precautions in contrast to methods using scintillation counting. Copyright © 2011 John Wiley & Sons, Ltd.     

Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience

5-Aminolaevulinic acid (ALA) is a precursor of protoporphyrin IX (Pp IX) in the biosynthetic pathway for haem. Certain types of cells have a large capacity to synthesize Pp IX when exposed to an adequate concentration of exogenous ALA. Since the conversion of Pp IX into haem is relatively slow, such cells tend to accumulate photosensitizing concentrations of Pp IX. Pp IX photosensitization can be induced in cells of the epidermis and its appendages, but not in the dermis. Moreover, since ALA in aqueous solution passes readily through abnormal keratin, but not through normal keratin, the topical application of ALA in aqueous solution to actinic keratoses or superficial basal cell or squamous cell carcinomas induces Pp IX photosensitization that is restricted primarily to the abnormal epithelium. Subsequent exposure to photoactivating light selectively destroys such lesions. In our ongoing clinical trial of ALA-induced Pp IX photodynamic therapy, the response rate for basal cell carcinomas following a single treatment has been 90% complete response and 7.5% partial response for the first 80 lesions treated. The cosmetic results have been excellent, and patient acceptance has been very good.     

Photodynamic therapy using 5-aminolaevulinic acid for oesophageal adenocarcinoma associated with Barrett's metaplasia

Photodynamic therapy (PDT) is a novel technique for local endoscopic treatment of gastrointestinal neoplasia. Current photosensitisers for PDT may cause prolonged skin phototoxicity. 5-Aminolaevulinic acid (ALA), a precursor of the photosensitiser protoporphyrin IX (PpIX), is more acceptable because of its short half-life and preferential accumulation in mucosa and mucosal tumour. We have treated 12 patients, median age 73 years (range 55-88) with oesophageal adenocarcinoma arising from Barrett's metaplasia (two carcinomas-in-situ, grade 0; 10 carcinomas, grade 1-11A based on endoluminal ultrasound in two and CT scanning in 10 patients). ALA (60 and 75 mg/kg body weight) was given orally in two or five equally divided doses. The PpIX distribution in stomach, normal oesophagus, Barrett's mucosa and carcinoma was measured by quantitative fluorescence photometry. PDT was performed using laser light (630 nm) delivered via a cylindrical diffuser 4-6 h after the first dose of ALA. The patients received one to four sessions of PDT. PpIX accumulation in the mucosa was two to three times that in the lamina propria. The differential distribution between carcinomatous and normal oesophageal mucosa was less marked (carcinoma:normal mucosa ratio = 1.4). Higher doses of ALA increased PpIX accumulation in all tissues but did not increase the differential PpIX distribution between tumour and normal oesophageal mucosa. After PDT using ALA (ALA/PDT), all mucosa showed superficial white necrotic changes and the histology confirmed fibrinoid necrosis. One patient with carcinoma-in-situ had the tumour eradicated after one treatment with no recurrence at 28 months. Another patient with a small T1 tumour required four ALA/PDT treatments, and died of other disease after 36 months. There was no evidence of recurrence. The tumour bulk in the other carcinomas was not significantly reduced. ALA/PDT has a potential for the eradication of small tumours but careful patient selection with endoluminal ultrasound is needed when using ALA/PDT to treat oesophageal cancer.