A potent nonporphyrin class of photodynamic therapeutic agent: Cellular localisation, cytotoxic potential and influence of hypoxia

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics. © 2005 Cancer Research UK.

In vitro demonstration of the heavy-atom effect for photodynamic therapy

Photodynamic therapy (PDT) is an emerging treatment modality for a range of disease classes, both cancerous and noncancerous. This has brought about an active pursuit of new PDT agents that can be optimized for the unique set of photophysical characteristics that are required for a successful clinical agent. We now describe a totally new class of PDT agent, the BF2-chelated 3,5-diaryl-1H-pyrrol-2-yl-3,5-diarylpyrrol-2-ylideneamines (tetraarylazadipyrromethenes). Optimized synthetic procedures have been developed to facilitate the generation of an array of specifically substituted derivatives to demonstrate how control of key therapeutic parameters such as wavelength of maximum absorbance and singlet-oxygen generation can be achieved. Photosensitizer absorption maxima can be varied within the body's therapeutic window between 650 and 700 nm, with high extinction coefficients ranging from 75,000 to 85,000 M(-1) cm(-1). Photosensitizer singlet-oxygen generation level was modulated by the exploitation of the heavy-atom effect. An array of photosensitizers with and without bromine atom substituents gave rise to a series of compounds with varying singlet-oxygen generation profiles. X-ray structural evidence indicates that the substitution of the bromine atoms has not caused a planarity distortion of the photosensitizer. Comparative singlet-oxygen production levels of each photosensitizer versus two standards demonstrated a modulating effect on singlet-oxygen generation depending upon substituent patterns about the photosensitizer. Confocal laser scanning microscopy imaging of 18a in HeLa cervical carcinoma cells proved that the photosensitizer was exclusively localized to the cellular cytoplasm. In vitro light-induced toxicity assays in HeLa cervical carcinoma and MRC5-SV40 transformed fibroblast cancer cell lines confirmed that the heavy-atom effect is viable in a live cellular system and that it can be exploited to modulate assay efficacy. Direct comparison of the efficacy of the photosensitizers 18b and 19b, which only differ in molecular structure by the presence of two bromine atoms, illustrated an increase in efficacy of more than a 1000-fold in both cell lines. All photosensitizers have very low to nondeterminable dark toxicity in our assay system.

Metal based photosensitizers of tetradentate Schiff base: Promising role in anti-tumor activity through singlet oxygen generation mechanism

In the present investigation, a Schiff base N'(1),N'(3)-bis(Z)-(2-hydroxynapthyl)methylidene]benzene-1,3-dicarbod ihydrazide (L-1) and its Co(II), Ni(II) and Cu(II) complexes have been synthesized and characterized as novel photosensitizing agents for photodynamic therapy (PDT). The interaction of these complexes with calf thymus DNA (CT DNA) has been explored using absorption, thermal denaturation and viscometric studies. The experimental results revealed that Co(II) and Ni(II) complexes on binding to CT DNA imply a covalent mode, most possibly involving guanine N7 nitrogen of DNA, with an intrinsic binding constant K-b of 4.5 x 10(4) M-1 and 4.2 x 10(4) M-1, respectively. However, interestingly, the Cu(II) complex is involved in the surface binding to minor groove via phosphate backbone of DNA double helix with an intrinsic binding constant K-b of 5.7 x 10(4) M-1. The Co(II), Ni(II) and Cu(II) complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to UV-visible light of 365 nm, through O-1(2) generation with quantum yields of 0.28, 0.25 and 0.30, respectively. Further, these complexes are cytotoxic in A549 lung cancer cells, showing an enhancement of cytotoxicity upon light irradiation. (C) 2013 Elsevier B.V. All rights reserved.

Avaliação da eficácia da terapia fotodinâmica como adjunto ao tratamento periodontal de pacientes com periodontite agressiva

A periodontite agressiva é um processo inflamatório de origem bacteriana mediado pelo sistema imunológico do hospedeiro e é provavelmente a forma mais grave de doença periodontal, apresentando destruição das estruturas tanto de proteção quanto de suporte dentário, num período relativamente rápido, normalmente levando a perda prematura dos elementos dentários e, em alguns casos, terapia antimicrobiana adjunta é necessária em adição à terapia mecânica. O objetivo do presente ensaio clínico randomizado controlado foi avaliar o efeito clínico da terapia fotodinâmica como adjunto ao tratamento periodontal não cirúrgico no tratamento da periodontite agressiva Os seguintes parâmetros clínicos foram avaliados: índice de placa visível, sangramento à sondagem, profundidade de bolsa à sondagem, nível de inserção clínica relativo, envolvimento de furca e mobilidade. Foram selecionados dez pacientes com periodontite agressiva, os quais foram examinados no dia zero e após três meses.O desenho do estudo consistiu em um modelo de boca dividida, onde um hemiarco foi tratado com raspagem e alisamento radicular e terapia fotodinâmica (laser diodo) e o outro apenas com raspagem e alisamento radicular. Três meses após o término do tratamento, os grupos terapêuticos apresentaram resultados semelhantes para todos os parâmetros clínicos avaliados: ambas as terapias tiveram sucesso, como redução de profundidade de bolsa, ganho de nível de inserção clínica relativo, redução de índice de placa visível, redução de sangramento à sondagem, diminuição de envolvimento de furca e diminuição de mobilidade, porém sem diferenças estatisticamente significantes entre elas. Dentro das limitações do presente estudo, os resultados sugerem que a terapia fotodinâmica adjuntamente ao tratamento periodontal não cirúrgico mecânico foi tão eficaz quanto o tratamento periodontal não cirúrgico mecânico sozinho.

Development of novel oral formulations prepared via hot melt extrusion for targeted delivery of photosensitizer to the colon

Colon-residing bacteria, such as vancomycin-resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit®-based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso-tetra(N-methyl-4-pyridyl)porphine tetra-tosylate (TMP), or 5-aminolevulinic acid hexyl-ester (h-ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen-releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6-h period. For TMP and h-ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon-residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.

Targeting of photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene using oligonucleotide-conjugates containing [Ru(phen)3](2+)-like photosensitiser groups

Photooxidative damage was induced predominantly at a single guanine base in a target DNA by irradiation (lambda > 330 nm) in the presence of complementary oligodeoxynucleotide conjugates (ODN-5'-linker-[Ru(phen)3]2+) (phen = 1,10-phenanthroline). The target DNA represents the b2a2 variant of the chimeric bcr-abl gene implicated in the pathogenesis of chronic myeloid leukaemia, and the sequence of the 17mer ODN component of the conjugate (3' G G T A G T T A T T C C T T C T T 5') was complementary to the junction region of the sense strand sequence of this oncogene. Two different conjugates were prepared, both of them by reaction of the appropriate succinimide ester with 5'-hexylamino-derivatised 17mer ODN. In Ru-ODN-1 (7) the linker was -(CH2)6-NHCO-bpyMe (-bpyMe = 4'-[4-methyl-2,2'-bipyridyl]), whereas in Ru-ODN-2 (13) it was -(CH2)6-NHCO-(CH2)3-CONH-phen. Photoexcitation of either of the conjugates when hybridised with the 32P-5'-end-labelled target 34mer 5'T G A C C A T C A A T A A G G A A G A A G21 C C C T T C A G C G G C C 3' (ODN binding site underlined) led to an alkali-labile site predominantly (> 90%) at the G21 base, which is at the junction of double-stranded and single-stranded regions of the hybrid. Greater yields were found with Ru-ODN-1 (7) than with Ru ODN-2 (13). In contrast to this specific cleavage with Ru-ODN-1 (7) or Ru-ODN-2 (13), alkali-labile sites were generated at all guanines when the 34mer was photolysed in the presence of the free sensitiser [Ru(phen)3]2+. Since [Ru(phen)3]2+ was shown to react with 2'-deoxyguanosine to form the diastereomers of a spiroiminodihydantoin derivative (the product from 1O2 reaction), 1O2 might also be an oxidizing species in the case of Ru-ODN-1 (7) and Ru-ODN-2 (13). Therefore to determine the range of reaction, a series of 'variant' targets was prepared, in which G21 was replaced with a cytosine and a guanine substituted for a base further towards the 3'-end (e.g. Variant 3; 5'T G A C C A T C A A T A A G G A A G A A C C G23 C T T C A G C G G32 C C3'). While it was noted that efficient reaction took place at distances apparently remote from the photosensitiser (e.g at G32, but not G23 for Variant 3), this effect could be attributed to hairpinning of the single-stranded region of the target. These results are therefore consistent with the photooxidative damage being induced by a reaction close to the photosensitiser rather than by a diffusible species such as 1O2.

Long-Term Recurrence of Nonmelanoma Skin Cancer after Topical Methylaminolevulinate Photodynamic Therapy in a Dermato-Oncology Department

BACKGROUND: Most available studies on the efficacy of topical photodynamic therapy focus on short-to medium-term results. Long-term data are scarce. OBJECTIVE: To evaluate the long-term efficacy of photodynamic therapy with topical methylaminolevulinate to treat Bowen's disease and basal cell carcinoma in the clinical practice setting of a dermato-oncology department. METHODS: The study included patients diagnosed with Bowen's disease or basal cell carcinoma, and who received photodynamic therapy from 2004 to 2008. Treatment protocol and clinical follow-up were standardized. The primary endpoint was clinically observed recurrence in a previous photodynamic therapy-treated area. Descriptive and survival analyses were performed. RESULTS: A total of 31 Bowen's disease lesions and 44 superficial basal cell carcinoma were treated, with a median follow-up of 43.5 months. Recurrence was observed in 14 Bowen's disease lesions (53.8%) and in 11 superficial basal cell carcinoma (33.3%). Significantly higher estimates for recurrence rates were found in patients with Bowen's disease (p=0.0036) or those aged under 58 years (p=0.039). The risk of recurrence was higher in patients with Bowen's disease than in those with superficial basal cell carcinoma and younger patients. CONCLUSIONS: Recurrence should be considered when choosing to treat non-melanoma skin cancer with photodynamic therapy. Younger age and Bowen's disease were independent predictors for long-term recurrence, suggesting the need to establish an extended period of follow-up for this subset of patients.

Antimicrobial Photodynamic Therapy in the Non-Surgical Treatment of Aggressive Periodontitis: Cytokine Profile in Gingival Crevicular Fluid, Preliminary Results

Background: Aggressive periodontitis is a specific form of periodontal disease that is characterized by rapid attachment loss and bone destruction. Cytokine profiles are of considerable value when studying disease course during treatment. The aim of this trial was to investigate cytokine levels in the gingival crevicular fluid (GCF) of patients with aggressive periodontitis, after treatment with photodynamic therapy (PDT) or scaling and root planing (SRP), in a split-mouth design on -7, 0, +1, +7, +30, and +90 days. Methods: Ten patients were randomly treated with PDT using a laser source associated with a photosensitizer or SRP with hand instruments. GCF samples were collected, and the concentrations of tumor necrosis factor-alpha (TNF-alpha) and receptor activator of nuclear factor-kappa B ligand (RANKL) were determined by enzyme-linked immunosorbent assays. The data were analyzed using generalized estimating equations to test the associations among treatments, evaluated parameters, and experimental times (alpha = 0.05). Results: Non-surgical periodontal treatment with PDT or SRP led to statistically significant reductions in TNF-alpha level 30 days following treatment. There were similar levels of TNF-alpha and RANKL at the different time points in both groups, with no statistically significant differences. Conclusion: SRP and PDT had similar effects on crevicular TNF-alpha and RANKL levels in patients with aggressive periodontitis. J Periodontol 2009;80:98-105.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

Treatment of ligature-induced peri-implantitis by lethal photosensitization and guided bone regeneration: A preliminary histologic study in dogs

Background: the purpose of this pilot study was to evaluate the healing potential and reosseointegration in ligature-induced peri-implantitis defects adjacent to various dental implant surfaces following lethal photosensitization.Methods: A total of 36 dental implants with 4 different surface coatings (9 commercially pure titanium surface [CPTi]; 9 titanium plasma-sprayed [TPS]; 9 hydroxyapatite [HA]; and 9 acid-etched [AE]) were inserted in 6 male mongrel dogs 3 months after extraction of mandibular premolars. After a 2-month period of ligature-induced peri-implantitis and 12 months of natural peri-implantitis progression, only 19 dental implants remained. The dogs underwent surgical debridement of the remaining dental implant sites and lethal photosensitization by combination of toluidine blue O (100 mug/ml) and irradiation with diode laser. All exposed dental implant surfaces and bone craters were meticulously cleaned by mechanical means, submitted to photodynamic therapy, and guided bone regeneration (GBR) using expanded polytetrafluoroethylene (ePTFE) membranes. Five months later, biopsies of the implant sites were dissected and prepared for ground sectioning and analysis.Results: the percentage of bone fill was HA: 48.28 +/- 15.00; TPS: 39.54 +/- 12.34; AE: 26.88 +/- 22.16; and CPTi: 26.70 +/- 16.50. The percentage of reosseointegration was TPS: 25.25 +/- 11.96; CPTi: 24.91 +/- 17.78; AE: 17.30 +/- 15.41; and HA: 15.83 +/- 9.64.Conclusion: These data suggest that lethal photosensitization may have potential in the treatment of peri-implantitis.

Lethal photosensitization in microbiological treatment of ligature-induced peri-implantitis: a preliminary study in dogs

This pilot study evaluated, by culture testing, the effectiveness of lethal photosensitization for the microbiological treatment of peri-implantitis in dogs. Experimental peri-implantitis was induced by ligature placement for 2 months. Following ligature removal, plaque control was instituted by scrubbing with 0.12% chlorhexidine daily for 12 months. Subsequently, mucoperiosteal flaps were elevated for scaling the implant surface. Microbial samples were obtained with paper points before and after treatment of implant surfaces by means of 100 microg/ml toluidine blue O (TBO,) and were exposed, for 80 s, to light with a wavelength of 685 nm from a 50 mW GaAlAs diode laser. The mean initial and final bacterial counts were 7.22 +/- 0.20 and 6.84 +/- 0.44 CFU/ml, respectively for TVC (P < 0.0001); 6.19 +/- 0.45 and 3.14 +/- 3.29 CFU/ml for P. intermedia/nigrescens (P = 0.001); 5.98 +/- 0.38 and 1.69 +/- 2.90 CFU/ml for Fusobacterium spp. (P = 0.001); and 6.07 +/- 0.22 to 1.69 +/- 2.94 CFU/ml for beta-hemolytic Streptococcus (P = 0.0039). It may be concluded that lethal photosensitization resulted in a reduction of the bacterial count. Complete elimination of bacteria was achieved in some samples.

Susceptibility of planktonic cultures of Streptococcus mutans to photodynamic therapy with a light-emitting diode

The objective of this study was to evaluate the effect of photodynamic therapy with erythrosine and rose bengal using a light-emitting diode (LED) on planktonic cultures of S. mutans. Ten S. mutans strains, including nine clinical strains and one reference strain (ATCC 35688), were used. Suspensions containing 10 6 cells/mL were prepared for each strain and were tested under different experimental conditions: a) LED irradiation in the presence of rose bengal as a photosensitizer (RB+L+); b) LED irradiation in the presence of erythrosine as a photosensitizer (E+L+); c) LED irradiation only (P-L+); d) treatment with rose bengal only (RB+L-); e) treatment with erythrosine only (E+L-); and f) no LED irradiation or photosensitizer treatment, which served as a control group (P-L-). After treatment, the strains were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were submitted to analysis of variance and the Tukey test (p ≤ 0.05). The number of CFU/mL was significantly lower in the groups submitted to photodynamic therapy (RB+L+ and E+L+) compared to control (P-L-), with a reduction of 6.86 log 10 in the RB+L+ group and of 5.16 log 10 in the E+L+ group. Photodynamic therapy with rose bengal and erythrosine exerted an antimicrobial effect on all S. mutans strains studied.

Photodynamic therapy for pythiosis

Background -  Pythiosis is a life-threatening disease caused by Pythium insidiosum. Photodynamic therapy (PDT) is an alternative treatment to surgery that uses the interaction of a photosensitizer, light and molecular oxygen to cause cell death. Objectives -  To evaluate the effect of PDT on the in vitro growth of P. insidiosum and in an in vivo model of pythiosis. Methods -  For in vitro studies, two photosensitizers were evaluated: a haematoporphyrin derivative (Photogem®) and a chlorine (Photodithazine®). AmphotericinB was also evaluated, and the control group was treated with sterile saline solution. All experiments (PDT, porphyrin, chlorine and light alone, amphotericinB and saline solution) were performed as five replicates. For in vivo studies, six rabbits were inoculated with 20,000 zoospores of P. insidiosum, and an area of 1cm3 was treated using the same sensitizers. The PDT irradiation was performed using a laser emitting at 660nm and a fluence of 200J/cm2. Rabbits were clinically evaluated daily and histopathological analysis was performed 72h after PDT. Results -  For in vitro assays, inhibition rates for PDT ranged from 60 to 100% and showed better results in comparison to amphotericinB. For the in vivo assays, after PDT, histological analysis of lesions showed a lack of infection up to 1cm in depth. Conclusions and clinical importance -  In vitro and in vivo studies showed that PDT was effective in the inactivation of P. insidiosum and may represent a new approach to treating pythiosis. © 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.

Dentin hypersensitivity after teeth bleaching with in-office systems. randomized clinical trial

Purpose: To comparatively and prospectively compare in a randomized clinical trial, dentin hypersensitivity after treatment with three in-office bleaching systems, based on hydrogen peroxide at different concentrations, with and without light source activation. Methods: 88 individuals were included according to inclusion and exclusion criteria. Subjects were randomly divided into the following three treatment groups: Group 1 was treated with three 15-minute applications of hydrogen peroxide at 15% with titanium dioxide (Lase Peroxide Lite) that was light-activated (Light Plus Whitening Lase) with five cycles of 1 minute and 30 seconds each cycle, giving a total treatment time of 45 minutes; Group 2 was treated with three 10-minute applications of hydrogen peroxide at 35% (Lase Peroxide Sensy), activated by light (LPWL) same activation cycles than Group 1, with a total treatment time of 30 minutes; Group 3 was treated with only one application for 45 minutes of hydrogen peroxide at 35% (Whitegold Office) without light activation. Each subject underwent one session of bleaching on the anterior teeth according to the manufacturers' instructions. Dentin sensitivity was recorded with a visual analogue scale (VAS) at baseline, immediately after, and at 7 and 30 days after treatment using a stimulus of an evaporative blowing triple syringe for 3 seconds on the upper central incisors from a distance of 1 cm. A Kruskal-Wallis test followed by Mann-Whitney test was performed for statistical analysis. Results: All groups showed increased sensitivity immediately after treatment. Group 1 displayed less changes relative to baseline with no significant differences (P= 0.104). At 7 and 30 days after treatment, a comparison of VAS values indicated no significant differences between all groups (P= 0.598 and 0.489, respectively).

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.