Taking PDT into mainstream clinical practice
Author(s):
Stephen G. Bown
Show Abstract
Many individuals in the field are frustrated by the slow progress getting PDT established in mainstream clinical practice.
The five key reasons are:
1. Lack of adequate evidence of safety and efficacy and optimization of dosimetry. These are fundamental. The
number of randomized controlled studies is still small. For some cancer applications, it is difficult to get
patients to agree to be randomised, so different approaches must be taken. Anecdotal results are not acceptable
to sceptics and regulators.
2. The regulatory processes. The rules get more complex every day, but there is no choice, they must be met. The
full bureaucratic strength of the pharmaceutical industry is needed to address these issues.
3. Conservatism of the medical profession. Established physicians are reluctant to change practice, especially if it
means referring patients to different specialists.
4. Lack of education. It is amazing how few physicians have even heard of PDT and many that have, are
sceptical. The profile of PDT to both the medical profession and the general public needs to be raised
dramatically. Patient demand works wonders!
5. Money. Major investment is required to run clinical trials. Pharmaceutical companies may see PDT as a threat
(eg reduced market for chemotherapy agents). Licensed photosensitisers are expensive. Why not reduce the
price initially, to get the technique established and stimulate demand? PDT has the potential for enormous cost
savings for health service providers.
With appropriate motivation and resources these problems can be addressed. Possible routes forward will be suggested.
Photodynamic application in neurosurgery: present and future
Author(s):
Herwig Kostron
Show Abstract
Photodynamic techniques such as photodynamic diagnosis (PDD), fluorescence guided tumor resection (FGR) and
photodynamic therapy (PDT) are currently undergoing intensive clinical investigations as adjunctive treatment for
malignant brain tumours.
This review provides an overview on the current clinical data and trials as well as on photosensitisers, technical
developments and indications for photodynamic application in Neurosurgery. Furthermore new developments and
clinical significance of FGR for neurosurgery will be discussed. Over 1000 patients were enrolled in various clinical
phase I/II trials for PDT for malignant brain tumours. Despite various treatment protocols, variation of photosensitisers
and light dose there is a clear trend towards prolonging median survival after one single PDT as compared to
conventional therapeutic modalities. The median survival after PDT for primary glioblastoma multiforme WHO IV was
19 months and for recurrent GBM 9 months as compared to standard convential treatment which is 15 months and 3
months, respectively. FGR in combination with adjunctive radiation was significantly superior to standard surgical
resection followed by radiation. The combination of FGR/PDD and intraoperative PDT increased significantly survival
in recurrent glioblastoma patients.
The combination of PDD/ FGR and PDT offers an exciting approach to the treatment of malignant brain tumours "to
see and to treat." PDT was generally well tolerated and side effects consisted of occasionally increased intracranial
pressure and prolonged skin sensitivity against direct sunlight.
This review covers the current available data and draws the future potential of PDD and PDT for its application in
neurosurgery.
Identifying initial molecular targets of PDT: protein and lipid oxidation products
Author(s):
Nancy L. Oleinick;
Junhwan Kim;
Myriam E. Rodriguez;
Liang-yan Xue;
Malcolm E. Kenney;
Vernon E. Anderson
Show Abstract
Photodynamic Therapy (PDT) generates singlet oxygen (1O2) which oxidizes biomolecules in the immediate vicinity of
its formation. The phthalocyanine photosensitizer Pc 4 localizes to mitochondria and endoplasmic reticulum, and the
primary targets of Pc 4-PDT are expected to be lipids and proteins of those membranes. The initial damage then causes
apoptosis in cancer cells via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by the
activation of caspases. That damage also triggers the induction of autophagy, an attempt by the cells to eliminate
damaged organelles, or when damage is too extensive, to promote cell death. Cyt-c is bound to the cytosolic side of the
mitochondrial inner membrane through association with cardiolipin (CL), a phospholipid containing four unsaturated
fatty acids and thus easily oxidized by 1O2 or by other oxidizing agents. Increasing evidence suggests that oxidation of
CL loosens its association with Cyt-c, and that the peroxidase activity of Cyt-c can oxidize CL. In earlier studies of Cyt-c
in homogeneous medium by MALDI-TOF-MS and LC-ESI-MS, we showed that 1O2 generated by Pc 4-PDT oxidized
histidine, methionine, tryptophan, and unexpectedly phenylalanine but not tyrosine. Most of the oxidation products were
known to be formed by other oxidizing agents, such as hydroxyl radical, superoxide radical anion, and peroxynitrite.
However, two products of histidine were unique to 1O2 and may be useful for reporting the action of 1O2 in cells and
tissues. These products, as well as CL oxidation products, have now been identified in liposomes and mitochondria after
Pc 4-PDT. In mitochondria, the PDT dose-dependent oxidations can be related to specific changes in mitochondrial
function, Bcl-2 photodamage, and Cyt-c release. Thus, the role of PDT-generated 1O2 in oxidizing Cyt-c and CL and the
interplay between protein and lipid targets may be highly relevant to understanding one mechanism for cell killing by
PDT.
Photodynamic therapy: first responses
Author(s):
David Kessel;
Michael Price
Show Abstract
During the irradiation of photosensitized cells, reactive oxygen species (ROS) are generated leading to a variety of effects
including apoptosis and autophagy. These responses can occur within minutes after irradiation. Apoptosis is an irreversible
pathway to death that can be triggered by release of cytochrome c from mitochondria. Autophagy is a recycling process that
can occur as a result of Bcl-2 photodamage or as a response to organelle disruption. We have reported that autophagy is
associated with a 'shoulder' on the PDT dose-response curve. Although predominantly a survival pathway, autophagy can
also play a role in cell death if cells attempt an excessive amount of recycling, beyond their ability to repair photodamage.
Recent studies have been directed toward assessing the role of different ROS in the immediate response to PDT. While
singlet oxygen is considered to be the major phototoxic ROS, it appears that catalase activity is also a determinant of the
apoptotic response and that H2O2•OH can amplify the effects of singlet oxygen. An early response to PDT also involves
inhibition of membrane trafficking systems related to the endocytic pathway. The extent and nature of these early responses
appear to be among the determinants of subsequent tumor eradication.
Signaling from lysosomes enhances mitochondria-mediated photodynamic therapy in cancer cells
Author(s):
Geraldine Quiogue;
Shalini Saggu;
Hsin-I Hung;
Malcolm E. Kenney;
Nancy L. Oleinick;
John J. Lemasters;
Anna-Liisa Nieminen
Show Abstract
In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in
singlet oxygen formation and cell death. Assessed by confocal microscopy, the photosensitizer phthalocyanine 4 (Pc 4)
localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. A Pc 4
derivative, Pc 181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 was a more effective photosensitizer
promoting killing cancer cells after PDT. The mode of cell death after Pc 181-PDT is predominantly apoptosis, and
pancaspase and caspase-3 inhibitors prevent onset of the cell death. To assess further how lysosomes contribute to PDT,
we monitored cell killing of A431cells after PDT in the presence and absence of bafilomycin, an inhibitor of the acidic
vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself
did not induce toxicity but greatly enhanced Pc 4-PDT-induced cell killing. In comparison to Pc 4, less enhancement of
cell killing by bafilomycin occurred after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the
absence of bafilomycin. These results indicate that lysosomal disruption can augment PDT with Pc 4, which targets
predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already targets lysosomes.
Anti-tumor immune response after photodynamic therapy
Author(s):
Pawel Mroz;
Ana P Castano;
Mei X Wu;
Andrew L Kung;
Michael R. Hamblin
Show Abstract
Anti-tumor immunity is stimulated after PDT due a number of factors including: the acute inflammatory response caused
by PDT, release of antigens from PDT-damaged tumor cells, priming of the adaptive immune system to recognize
tumor-associated antigens (TAA), and induction of heat-shock proteins. The induction of specific CD8+ T-lymphocyte
cells that recognize major histocompatibility complex class I (MHC-I) restricted epitopes of TAAs is a highly desirable
goal in cancer therapy as it would allow the treatment of tumors that may have already metastasized. The PDT killed
tumor cells may be phagocytosed by dendritic cells (DC) that then migrate to draining lymph nodes and prime naïve T-cells
that recognize TAA epitopes. We have carried out in vivo PDT with a BPD-mediated vascular regimen using a pair
of BALB/c mouse colon carcinomas: CT26 wild type expressing the naturally occurring retroviral antigen gp70 and
CT26.CL25 additionally expressing beta-galactosidase (b-gal) as a model tumor rejection antigen. PDT of CT26.CL25
cured 100% of tumors but none of the CT26WT tumors (all recurred). Cured CT26.CL25 mice were resistant to
rechallenge. Moreover mice with two bilateral CT26.CL25 tumors that had only one treated with PDT demonstrated
spontaneous regression of 70% of untreated contralateral tumors. T-lymphocytes were isolated from lymph nodes of
PDT cured mice that recognized a particular peptide specific to b-gal antigen. T-lymphocytes from LN were able to kill
CT26.CL25 target cells in vitro but not CT26WT cells as shown by a chromium release assay. CT26.CL25 tumors
treated with PDT and removed five days later had higher levels of Th1 cytokines than CT26 WT tumors showing a
higher level of immune response. When mice bearing CT26WT tumors were treated with a regimen of low dose
cyclophosphamide (CY) 2 days before, PDT led to 100% of cures (versus 0% without CY) and resistance to rechallenge.
Low dose CY is thought to deplete regulatory T-cells (Treg, CD4+CD25+foxp3+) and potentiate immune response after
PDT in the case of tumors that express self-antigens. These data suggest that PDT alone will stimulate a strong immune
response when tumors express a robust antigen, and in cases where tumors express a self-antigen, T-reg depletion can
unmask the immune response after PDT.
Combination of PDT and a DNA demethylating agent produces anti-tumor immune response in a mouse tumor model
Author(s):
Pawel Mroz;
Michael R. Hamblin
Show Abstract
Epigenetic mechanisms, which involve DNA methylation and histone modifications, result in the heritable silencing of
genes without a change in their coding sequence. However, these changes must be actively maintained after each cell
division rendering them a promising target for pharmacologic inhibition. DNA methyltransferase inhibitors like 5-aza-deoxycytidine
(5-aza-dC) induce and/or up-regulate the expression of MAGE-type antigens in human and mice cancer
cells. Photodynamic therapy (PDT) has been shown to be an effective locally ablative anti-cancer treatment that has the
additional advantage of stimulating tumor-directed immune response. We studied the effects of a new therapy that
combined the demethylating agent 5-aza-dC with PDT in the breast cancer model 4T1 syngenic to immunocompetent
BALB/c mice. PDT was used as a locally ablating tumor treatment that is capable of eliciting strong and tumor directed
immune response while 5-aza-dC pretreatment was used promote de novo induction of the expression of P1A.protein.
This is the mouse homolog of human MAGE family antigens and is reported to function as a tumor rejection antigen in
certain mouse tumors. This strategy led to an increase in PDT-mediated immune response and better treatment outcome.
These results strongly suggest that the MAGE family antigens are important target for PDT mediated immune response
but that their expression can be silenced by epigenetic mechanisms. Therefore the possibility that PDT can be combined
with epigenetic strategies to elicit anti-tumor immunity in MAGE-positive tumor models is highly clinically significant
and should be studied in detail.
Intravital fiber-optic fluorescence imaging for monitoring ovarian carcinoma progression and treatment response
Author(s):
Bryan Q. Spring;
Jonathan P. Celli;
Conor L Evans;
Wei Zhong;
Imran Rizvi;
Zhiming Mai;
Jerome Mertz;
Seok H. Yun;
Tayyaba Hasan
Show Abstract
Our laboratory has constructed a custom fluorescence microendoscope for detecting and monitoring tumor nodules in a
mouse model of metastatic ovarian carcinoma (OVCA). The microendoscope is being applied for tumor recognition and
for quantifying tumor burden reduction following photodynamic therapy (PDT). Benzoporphyrin derivative monoacid
ring A (BPD-MA), a photosensitizing agent for PDT, is administered to the mice and imaged with the microendoscope
prior to PDT. BPD-MA fluorescence is a convenient means for locating tumor sites and quantifying tumor burden
(despite the fact that BPD-MA is a non-targeted contrast agent). The miniature, flexible microendoscope probe is
delivered via a 14-gauge catheter for imaging metastases along the outer surfaces of the internal organs and the inner
walls of the peritoneal cavity. The minimal invasiveness of this approach facilitates frequent imaging of the mice in
order to monitor cancer progression and treatment response. We present promising data for intravital imaging of
treatment response following PDT and new developments in the microendoscope instrumentation for improved image
quality.
Variables in photodynamic therapy for Barrett's esophagus
Author(s):
Linda R. Jones;
Norris W. Preyer;
Jane Buchanan;
Daryl M. Reynolds;
Herbert C. Wolfsen;
Michael B. Wallace;
Kanwar R. S. Gill
Show Abstract
Photodynamic therapy with porfimer sodium (PS) is a treatment option for high grade dysplasia associated with Barrett's
esophagus. This study sought to investigate the optical properties of Barrett's dysplasia that may be useful in light
dosimetry planning and to determine the effect of PS on tissue absorption and scattering. Fiber optic reflectance spectra
were collected before and 48 hours after administration of 2 mg/kg PS. Mucosal biopsies were collected at the same
locations. According to Monte Carlo analysis, the fiber optic probe sampled only the mucosal layer. A mathematical fit
of the reflectance spectra was performed as a function of blood volume fraction, oxygen saturation and scattering. The
average calculated blood volume was 100% higher in Barrett's tissue than normal esophageal tissue. The average
scattering slope from 620 to 750 nm was 26% higher for Barrett's dysplasia than normal esophageal tissue, indicating an
increase in the size of scattering particles. The difference in the scattering amplitude was not statistically significant,
suggesting no significant increase in the number of scattering particles. PS tissue content was determined with extraction
methods. Changes in the scattering slope due to PS sensitization were observed; however they were not proportional to
the extracted PS concentration.
Photoreactions for total antioxidant status measurement in humans
Author(s):
David Olivier;
Samuel Douillard;
Isabelle Lhommeau;
Edith Bigot;
Thierry Patrice
Show Abstract
Type 2 photoreactions are singlet oxygen (1O2) mediated. This singlet oxygen will de activate by reacting with a number
of targets and produce ROS and peroxides that will in turn give Reactive Oxygen Species (ROS). In order to verify
whether sera had the same capability to do so we provoked a photoreaction using Rose Bengal (RB) added to sera of 53
healthy donors and at the end of light delivery 2' -7' dichlorofluorescin (DCFH) had been added. In order to avoid any
optical artefact we analysed the influence of haemolysis on the fluorescent 2' -7' dichlorofluorescin (DCF) induced by
oxidising species. Reactivity to singlet oxygen was slightly different between men and women, varied slightly with age
and platelet number. It was remarkably stable over a large period of time and thus represents a characteristic of each
serum. Studies are in progress to verify whether ROS dependent diseases would impair this reactivity to singlet oxygen.
Efficacy of low-dose mTHPC-PDT for the treatment of basal cell carcinomas
Author(s):
Christian S. Betz;
Winrich Rauschning;
Evgueni P. Stranadko;
Mikhail V. Riabov;
Volker Albrecht;
Nikolay E. Nifantiev;
Colin Hopper
Show Abstract
Objectives: Basal cell carcinomas (BCCs) are the most common skin cancers, and incidence rates are still rising.
Photodynamic Therapy (PDT) with mTHPC (Foscan®) has shown to be a promising alternative to other treatments with
good cosmetic results. This study was performed to determine optimal treatment parameters for this indication.
Methods: 117 patients with a total of 460 BCCs received mTHPC-PDT. The treatment parameters were varied as
follows: Foscan® dose 0.03 - 0.15 mg/kg, drug-light interval (DLI) 1 - 96 hours, total energy density 20 - 120 J/cm2.
The clinical outcomes were assessed 8 weeks after PDT following WHO guidelines.
Results: The rate of complete remissions (CR) was 96.7% and the general cosmetic outcome rated very good. In the
largest subgroup (n=80) with low-dose mTHPC (0.05 mg/kg mTHPC; 48 hours DLI; 50 J/cm2 total energy density), a
CR rate of 100% was accomplished. Minor changes of the parameters (0.04 mg/kg mTHPC or 24 hours DLI) yielded
similar results. Side effects were encountered in 52 out of 133 PDT sessions. They were more common in patients who
had received high drug doses (0.06 - 0.15 mg/kg) and comprised pain and phototoxic reactions. 3 patients developed
severe sunburns with subsequent scarring at the injection site following sunlight exposure 2-3 weeks after mTHPC
administration.
Conclusions: The data suggests that low-dose mTHPC-PDT is an effective treatment option for BCCs. If sensibly
applied, it is well tolerated and provides mostly excellent cosmetic results. The evaluation of long term results is still to
be undertaken.
Which intracranial lesions would be suitable for fluoresce guided resection?: A prospective review of 110 consecutive lesions
Author(s):
M. Sam Eljamel
Show Abstract
Fluorescence guided surgical resection is based on the basic principle that
photosensitizers are preferentially taken up and retained by cancer cells and the these
sensitizers absorb light in one band of the light spectrum and release the energy as
fluorescence that can be visualised in a different band of the spectrum. This study reports
fluorescence outcome of 110 consecutive intracranial tumors to identify its potential
benefits during fluorescence guided resection (FGR). The specificity of this technique
was very high with no false positive results. Its sensitivity varied from 83.7% in brain
lung metastasis to 85.7% in glioblastomas. A wide range of other intracranial tumors
were also studied but the numbers in each of these subgroups were small to deduct its
usefulness. However, grade III astrocytomas, primary cerebral lymphoma and
meningiomas fluoresced very well in all patients. FGR would be therefore very useful aid
during a wide range of intracranial tumor surgery with the advantages of specificity, high
sensitivity and would not be affected by brain shift and tumor removal. The limitations of
this technique were that the sensitivity was not 100% and it would be possible to miss 15% of tumors, blood can also obscure the fluorescence and it would be a problem in
vascular tumors, and photo bleaching can reduce the fluorescence.
Palliative photodynamic therapy for biliary tract carcinoma may improve survival and has a similar outcome to attempted curative surgery with positive resection margins
Author(s):
Stephen P. Pereira;
W. Rudiger Matull;
Dipok K. Dhar;
Laskshmana Ayaru;
Neomal S. Sandanayake;
Michael H. Chapman
Show Abstract
There is a need for better management strategies to improve survival and quality of life in patients with biliary tract
cancer (BTC). We compared treatment outcomes in 321 patients (median age 65 years, range 29-102; F:M; 1:1) with a
final diagnosis of BTC (cholangiocarcinoma n=237, gallbladder cancer n=84) seen in a tertiary referral cancer centre
between 1998-2007. Of 89 (28%) patients who underwent surgical intervention with curative intent, 38% had R0
resections and had the most favourable outcome, with a 3 year survival of 57%. Even though PDT patients had more
advanced clinical T-stages, their survival was similar to those treated with attempted curative surgery which resulted in
R1/2 resections (median survival 12 vs. 13 months, ns). In a subgroup of 36 patients with locally advanced BTC treated
with PDT as part of a prospective phase II study, the median survival was 12 (range 2-51) months, compared with 5
months in matched historical controls treated with stenting alone (p < 0.0001). In this large UK series, long-term survival
with BTC was only achieved in surgical patients with R0 resection margins. Palliative PDT resulted in similar survival to
those with curatively intended R1/R2 resections.
Nanotechnology-based combination therapy improves treatment response in cancer models
Author(s):
Prakash Rai;
Sung K. Chang;
Zhiming Mai;
Daniel Neuman;
Tayyaba Hasan
Show Abstract
Pancreatic cancer (PanCa) has a poor prognosis with a 5-year survival rate of only 5%. Photodynamic therapy (PDT) has
shown promising results in treating PanCa. Mechanism-based combinations with PDT have enhanced treatment
outcome. Agents tested with PDT include Avastin, an antibody against vascular endothelial growth factor (VEGF) which
is approved for treating various cancers. Simultaneous delivery of drugs in nano-constructs could improve the treatment
response of mechanism based combination therapies. Here, we investigate the effect of neutralizing VEGF using
nanotechnology for the delivery of Avastin in combination with PDT. For this we used a construct called "nanocells" in
which the photosensitizer was trapped inside polymer nanoparticles and these, with Avastin, were then encapsulated
inside liposomes. In vitro, nanocells containing Avastin (NCA) significantly enhanced cytotoxicity in PanCa cells. NCA
based PDT also significantly improved treatment response in mice that were orthotopically implanted with PanCa.
Avastin delivered extracellularly in combination with PDT did not show any improvement. Here we propose a new
paradigm for Avastin-based therapy by combining intracellular delivery of the antibody and PDT using nanotechnology
for treating PanCa.
Apoptotic induction by photodynamic therapy using hexaminolevulinate with a literature review
Author(s):
Ingegerd E. Furre;
Jahn M. Nesland;
Qian Peng
Show Abstract
Since the first report by Agarwal et al in 1991 on apoptotic induction by photodynamic therapy (PDT) with chloroaluminium phthalocyanine a large number of papers have come out to show that PDT can induce cell death through apoptosis. This finding may provide potential clinical impact on, for example, those tumor cells
resistant to any cancer therapy. The present paper briefly reviews apoptosis with emphasis on PDT-induced apoptosis with hexaminolevulinate.
Novel multifunctional acyloxyalkyl ester prodrugs of 5-aminolevulinic acid display improved anticancer activity dependent on photoactivation
Author(s):
G. Berkovitch;
A. Nudelman;
B. Ehenberg;
A. Rephaeli;
Z. Malik
Show Abstract
New approaches to PDT using multifunctional 5-aminolevulinic acid (ALA) based prodrugs activating mutual routes of
toxicity are described. We investigated the mutual anti-cancer activity of ALA prodrugs which upon metabolic
hydrolysis by unspecific esterases release ALA, formaldehyde or acetaldehye and the histone deacetylase inhibitor
(HDACI) butyric acid. The most potent prodrug in this study was butyryloxyethyl 5-amino-4-oxopentanoate (AN-233)
that stimulated a rapid biosynthesis of protoporphyrin IX (PpIX) in human glioblastoma U-251 cells and generated an
efficient photodynamic destruction. AN-233 induced a considerable high level of intracellular ROS in the cells following
light irradiation, reduction of mitochondrial activity, dissipation of the mitochondrial membrane potential resulting in
necrotic and apoptotic cell death. The main advantage of AN-233 over ALA stems from its ability to induce photodamage
at a significantly lower dose than ALA.
Enhancing protoporphyrin IX-induced PDT
Author(s):
Alison Curnow;
Andrew Pye;
Sandra Campbell
Show Abstract
Photodynamic therapy (PDT) using porphyrin precursors is commonly used in dermatology. Evidence indicates that
good clinical outcomes (associated with excellent cosmesis) can be achieved in superficial precancers and basal cell
carcinoma (BCC), however, efficacy appears less favorable for thicker nodular BCC (nBCC) unless multiple PDT
treatment cycles are performed. Enhancement is therefore required if nBCC lesions are to be treated effectively with a
single PDT treatment. The most common technique currently being routinely employed clinically is the use of
aminolevulinic acid (ALA) esters (usually methyl (MAL) or hexyl (HAL)). Standard dermatological PDT employing
these porphyrin precursors already manipulates the normal heme biosynthesis pathway resulting in a temporary
accumulation of the natural photosensitizer, protoporphyrin IX (PpIX). Further manipulation using iron chelating agents
is possible however. In normal and malignant human cells in vitro, the novel iron chelating agent CP94 produced greater
PPIX fluorescence when administered with ALA or MAL than either congener produced alone. CP94 was also
significantly more effective than the clinically established iron chelating agent desferrioxamine (DFO). Topical
application of ALA+CP94 to clinical nBCC lesions was a simple and safe treatment modification which produced a
significant increase in clinical clearance when CP94 was included in the cream.
The effect of ALA/PpIX PDT on putative cancer stem cells in tumor side populations
Author(s):
Janet Morgan;
Cara M. Petrucci
Show Abstract
Protoporphyrin IX (PpIX) synthesized endogenously from 5-aminolevulinic acid (ALA), is effluxed from cells
expressing the ATP-dependent transporter ABCG2. Side population (SP) cells (named for their low red/blue
fluorescence distribution in flow cytometry plots with ABCG2 substrates such as Hoechst) are postulated to contain
cancer stem cells (CSC). The SP in U87 (human gliblastoma cell line) were more resistant to ALA-PDT than NON-SP
cells. Inhibiting ABCG2 activity with the tyrosine kinase inhibitor imatinib mesylate (IM, Gleevec) during incubation
with ALA increased PpIX in the SP by preventing its efflux and decreased the SP after subsequent PDT, enhancing
phototoxicity. Evasion of SP cells from ALA-PDT could cause tumor recurrence from CSC. Manipulation of ABCG2
levels on the SP with small molecule modulators may be a potential strategy for enhancing PDT by decreasing the
amount of substrate photosensitizer extruded from cells and lowering the threshold for phototoxicity.
Novel drug delivery strategies for porphyrins and porphyrin precursors
Author(s):
D. I. J. Morrow;
R. F. Donnelly
Show Abstract
superficial lesions, such as actinic keratosis. In addition, photodynamic antimicrobial chemotherapy (PACT) is
attracting increasing interest for the treatment of infection. However, delivery strategies for topical PDT and PACT
are still based on application of rather simplistic cream and solution formulations, with little consideration given to
thermodynamics, targeting or the physicochemical properties of the active agent.
Purpose-designed dosage forms for topical delivery of aminolevulinic acid or its esters include creams
containing penetration enhancers and/or iron chelators, pressure sensitive patches and bioadhesive patches. Such
systems aim to enhance drug delivery across the stratum corneum and keratinised debris overlying neoplastic lesions
and improve subsequent protoporphyrin IX (PpIX) production.
The alternative to using porphyrin precursors is the use of pre-formed photosensitisers. However, owing to
their relatively high molecular weights, conventional topical application is not appropriate. Innovative strategies,
such as the use of needle-free injections and microneedle arrays, bypass the stratum corneum, enabling rapid and
targeted delivery not only porphyrin precursors but also pre-formed photosensitisers.
This presentation will review drug delivery work published to date in the fields of PDT and PACT. In
addition, the benefits of employing the latest advances in pharmaceutical technology will be highlighted.
ALA PDT for high grade dysplasia in Barrett's oesophagus: review of a decade's experience
Author(s):
Stephen G. Bown;
Gary D. Mackenzie;
Jason M. Dunn;
Sally M. Thorpe;
Laurence B. Lovat
Show Abstract
We have been investigating PDT with 5 aminolaevulinic acid (ALA) for the treatment of high grade dysplasia (HGD) in
Barrett's oesophagus (BO) for over a decade. This drug has inherent advantages over porfimer sodium (Photofrin), the
current approved photosensitiser in the UK and USA, which causes strictures in 18-50% and light sensitivity for up to
three months. ALA has a lower rate of oesophageal strictures due to its preferential activity in the mucosa, sparing the
underlying muscle, and patients are only light sensitive for 1-2 days.
Within a randomised controlled trial, we demonstrated that an ALA dose of 60mg/kg activated by 1000J/cm red laser
light is the most effective. Using these values we achieved complete reversal of HGD at 1 year in 89% of 27 patients.
A randomised controlled trial of ALA vs porfimer sodium PDT for HGD is currently under way with end points of
efficacy and safety. 50 of 66 patients have been recruited. Preliminary data suggest ALA PDT is safer with a trend to
higher efficacy.
Late relapse can occur in 20% of patients. New prognostic markers, in particular aneuploidy, are helping us to identify
and target patients at risk of late relapse. Furthermore optical biopsy techniques such as elastic scattering spectroscopy
(ESS) may allow detection of nuclear abnormalities in vivo and enable us to target areas of interest whilst reducing
sampling error.
PDT faces new challenges for the treatment of HGD in BO, with the recent introduction of balloon based radiofrequency
ablation. This technique appears simpler and as effective as PDT, but follow up is currently short and long term safety
data is lacking. In our experience ALA PDT is currently the most effective minimally invasive treatment for HGD in
BO.
This work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR
Biomedical Research Centres funding scheme.
Targeted opening of the blood brain barrier by ALA-mediated photodynamic therapy
Author(s):
Henry Hirschberg;
Qian Peng;
Francisco A. Uzal;
David Chighvinadze;
Michelle J. Zhang;
Steen J. Madsen
Show Abstract
Background and Objective: We have evaluated the ability of ALA-mediated PDT to
selectivly open the BBB in rats. This will permit access of chemotherapeutic agents to
brain tumor cells remaining in the resection cavity wall, but limit their penetration into
normal brain remote from the site of illumination.
Study Design/Materials and Methods: ALA-PDT was performed on non-tumor bearing
inbred Fischer rats at increasing fluence levels. Contrast T1-weighted high field (3 T)
magnetic resonance imaging (MRI) scans were used to monitor the degree of BBB
disruption.
Results: PDT at increasing fluence levels between 9 and 17 J demonstrated an increasing
contrast flow rate. The BBB was found to be disrupted 2 h following PDT and 80 - 100
% restored 72 h later at the lowest fluence level. No effect on the BBB was observed if
26 J of light was given in the absence of ALA.
Conclusion: ALA-PDT was highly effective in opening the BBB in a localized region of
the brain. The degradation of the BBB was temporary in nature at fluence levels of 9 J,
opening rapidly following treatment and significantly restored during the next 72 h. No
signs of permanent tissue damage were seen on histological sections at this fluence level.
ALA-mediated fluorescence-guided resection (FGR) and PDT of glioma
Author(s):
Ann Johansson;
Herbert Stepp;
Tobias Beck;
Wolfgang Beyer;
Thomas Pongratz;
Ronald Sroka;
Thomas Meinel;
Walter Stummer;
Friedrich-Wilhelm Kreth;
Jörg-Christian Tonn;
Reinhold Baumgartner
Show Abstract
A summary of clinical trials employing photodynamic diagnosis (PDD) and photodynamic therapy (PDT) for
the diagnosis and treatment of brain malignancies is presented. Intra-cavity PDT has been performed within
the surgical cavity following FGR, employing oral administration of 5-aminolevulinic acid (5-ALA), either targeting
fluorescing tissue regions that were not removed during FGR due to safety reasons (referred to as focal
PDT, n=20) or illuminating the entire resection cavity (referred to as integral PDT, n=9). Both approaches
proved technically feasible and safe. Spectroscopic measurements performed pre-, during and post-PDT revealed
Protoporphyrin IX (PpIX)-photobleaching of more than 95% after the delivery of 200 J/cm2. This light dose
did not induce any side effects. Furthermore, interstitial PDT (iPDT) has been employed within one feasibility
trial (n=10) and one Phase I/II trial (n=15). Here, three to six cylindrical light diffusors (20-30 mm length,
200 mW/cm, 720 J/cm) were positioned within the target tissue under stereotactic guidance. Pre-treatment
planning was performed with the intent to target the entire tumour volume with a sufficient light dose while
also minimising the risk of any light-induced temperature increase. For the feasibility trial patients with small,
recurrent gliomas were included, resulting in a median survival of 15 months as well as some unexpected longterm
survivals (up to 5 years). The Phase I/II trial employed the same clinical procedures. Here, the 12-month
survival was 35% and the median progression-free survival was 6 months. In summary, stereotactic iPDT in
combination with treatment-planning could be shown to be a safe and feasible treatment modality. These trials
are presently being extended to also include on-line monitoring of PpIX fluorescence and photobleaching kinetics.
Preliminary data has revealed dramatically different PpIX levels and photobleaching kinetics. Such data
could possibly be employed for realtime treatment monitoring and as an early prognostic marker for the PDT
response.
Novel PDD-PDT system based on spectrophotometric real-time fluorescence monitoring and MALDI-TOF-MS analysis of tumors
Author(s):
Takato O. Yoshida;
Eiji Kohno;
Marc Dodeller;
Takashi Sakurai;
Seiji Yamamoto;
Susumu Terakawa
Show Abstract
In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not
by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the
cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during Photofrin®-PDT and
ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence
spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of
tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was
monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence
spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral
analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of
three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in
the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors
showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of
4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a Photofrin® PDD-PDT monitoring
tumor.
In-vivo fluorescence dosimetry of aminolevulinate-based protoporphyrin IX (PpIX) accumulation in human nonmelanoma skin cancers and precancers
Author(s):
Christine B. Warren;
Sara Lohser;
Sung Chang;
Philip A. Bailin;
Edward V. Maytin
Show Abstract
PDT is clinically useful for precancers (actinic keratoses; AK) of the skin, but the optimal duration for 5-ALA
application is still controversial. For basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), cure rates remain
inferior to surgical excision. Lack of knowledge about regional levels of PpIX levels within target tissues clearly
contribute to these suboptimal results. To investigate PpIX levels achievable in human skin neoplasias in-vivo, a clinical
study to monitor PpIX accumulation in vivo was performed. PpIX-fluorescence in patients undergoing ALA-PDT for
facial AK was monitored via real-time in-vivo fluorescence dosimetry, with measurements q20 min following
application of 5-ALA (Levulan Kerastick). PpIX accumulation followed linear kinetics in nearly all cases. The slopes
varied widely, and did not correlate with clinical outcome in all patients. Some patients with a low accumulation of
PpIX fluorescence had a good response to therapy, whereas others with high PpIX accumulation required repeat
treatment (although not necessarily of the same lesion). PpIX accumulation rates did correlate to a certain degree with
the overall amount of erythema. We conclude that unknown factors besides PpIX levels must be critical for the response
to treatment. To assess the relationship between PpIX levels in various skin cancers, patients undergoing routine Mohs
surgery for BCC or SCC were measured by in-vivo dosimetry at 2 h after 5-ALA application. Overall, a progressive
increase in PpIX signal during malignant progression was observed, in the following rank order: Normal skin < AK <
SCC ~ BCC.
New pain-relieving strategies for topical photodynamic therapy
Author(s):
Christina B. Halldin;
John Paoli;
Carin Sandberg;
Marica B. Ericson;
Helena Gonzalez;
Ann-Marie Wennberg
Show Abstract
PDT is an effective method when treating multiple actinic keratoses (field cancerization). The major side effect is pain.
Our objectives were to investigate the pain-relieving effect of transcutaneous electrical nerve stimulation (TENS) and
peripheral nerve blocks during PDT of field cancerization (FC) of the face and scalp. Patients with field cancerization
were included in three studies. In the first study, we examined TENS with an application site on the adjacent dermatome
from the PDT area in order to allow the use of water spray during PDT for FC of the scalp and face. In the second study,
patients with FC in the facial area received unilateral supraorbital, infraorbital and/or mental nerve blocks. The non-anaesthetised
side of the treatment area served as control. In the third study, with similar methodology as in the second
study, occipital and supraorbital nerve blocks were combined for FC of the forehead and scalp. The results of the studies
strongly support the use of nerve blocks as pain relief during PDT. The use of TENS provided a limited pain reduction,
but TENS might be an alternative if the patient disapproves of the use of nerve blocks or is afraid of injections.
The utilization of a non-invasive fluorescence imaging system to follow clinical dermatological MAL-PDT
Author(s):
Jessica Tyrrell;
Sandra Campbell;
Alison Curnow
Show Abstract
This study employed a commercially available, non-invasive, fluorescence imaging system (Dyaderm, Biocam,
Germany), to measure protoporphyrin IX (PpIX) concentration at several different stages during clinical dermatological
methyl aminolevulinate photodynamic therapy (MAL-PDT). We validated the system prior to use to ensure that the
PpIX changes witnessed were accurate and not due to environmental or user induced artifacts. The system was then
employed to acquire color (morphological) and fluorescent (physiological) images simultaneously during dermatological
PDT. Clinical data was collected from a range of licensed dermatological conditions (actinic keratosis, Bowen's disease
and superficial basal cell carcinoma) during initial and subsequent PDT treatment cycles. The initial clinical data
indicated that each type of licensed lesion considered responded in a similar manner following the application of
Metvix (Galderma, U.K.) and the subsequent light irradiation (Aktilite, Galderma, U.K.). Images acquired three
hours after Metvix application showed a significant increase in PpIX concentration within the lesion (P < 0.05), whilst
PpIX levels in the surrounding normal tissue remained unaltered. After irradiation, the PpIX concentration was
significantly decreased and returned to a level similar to the initial concentration originally observed. Lesions that
received subsequent treatment cycles accumulated significantly less PpIX (P < 0.05) prior to irradiation.
Low dose Photofrin PDT for recurrent in-situ squamous cell tumors of the head and neck
Author(s):
R. R Allison;
C. Austerlitz;
C. Sheng;
H. Mota;
B. Brodish;
P. Camnitz;
C. H. Sibata
Show Abstract
Multifocal recurrence of in-situ squamous cell cancer of the oral cavity, pharynx and vocal cord
following surgical failure can be a therapeutic dilemma. Salvage surgery or radiation may be an option but
morbidity can be significant. We evaluated the potential role of low dose Photofrin (1.2mg/Kg)
Photodynamic Therapy for this cohort of patients.
A total of 25 patients with multifocal recurrent in-situ squamous cell cancer of the oral cavity,
pharynx and vocal cord who had failed local resection, and where additional surgery or radiation therapy
would likely result in permanent morbidity, were offered Photodynamic Therapy. PDT consisted of off
label infusion of Photofrin (1.2mg/kg) followed 48 hours later by illumination at 630nm employing a light
diffuser (300J) and/or microlens (150Jcm2).
All patients completed their prescribed PDT and no patient has been lost to follow up (minimum 1
year). No photosensitivity reactions were noted. No significant morbidity was seen. All patients were able
to maintain oral nutrition. Procedure related pain was well controlled by one week of oral narcotics. At
one month post PDT all patients were biopsy negative in the treatment region and no failures within the
treatment region have been noted. No fibrosis or permanent PDT morbidity has been seen with follow up to
three years. Vocal cord and voice function were excellent.
Three patients developed new regions of in-situ disease outside the PDT fields, two underwent additional
PDT and one had laser resection.
Low dose Photofrin PDT offers excellent palliation and durable local control of recurrent in-situ
squamous cell cancers of the oral cavity, pharynx and true cords. This is a well tolerated therapy. Low
dose Photofrin appears to improve selectivity and minimize normal tissue injury. It should be tested in a
larger patient population.
Photodynamic management of bladder cancer
Author(s):
A. Johansson;
H. Stepp;
W. Beyer;
T. Pongratz;
R. Sroka;
M. Bader;
M. Kriegmair;
D. Zaak;
R. Waidelich;
A. Karl;
A. Hofstetter;
C. Stief;
R. Baumgartner
Show Abstract
Bladder cancer (BC) is among the most expensive oncological diseases. Any improvement in diagnosis or therapy
carries a high potential for reducing costs. Fluorescence cystoscopy relies on a selective formation of Protoporphyrin IX
(PpIX) or more general photoactive porphyrins (PAP) in malignant urothelium upon instillation of 5-aminolevulinic acid
(5-ALA) or its hexyl-derivative h-ALA. Fluorescence cystoscopy equipment has been developed with the aim to
compensate for the undesired distortion caused by the tissue optical properties by displaying the red fluorescence
simultaneously with the backscattered blue light. Many clinical studies proved a high sensitivity in detecting flat
carcinoma in situ and small papillary malignant tumours. As a result, recurrence rates were significantly decreased in
most studies. The limitation lies in a low specificity, caused by false positive findings at inflamed bladder wall. Optical
coherence tomography (OCT) is currently being investigated as a promising tool to overcome this limitation.
H-ALA-PDT (8 or 16 mM h-ALA in 50 ml instillation for 1-2 h, white light source, catheter applicator) has recently
been investigated in a phase I study. 17 patients were applied 100 J/cm2 (3 patients received incrementing doses of 25 -
50 - 100 J/cm2) during approx. 1 hour irradiation time in 3 sessions, 6 weeks apart. PDT was performed without any
technical complications. Complete photobleaching of the PpIX-fluorescence, as intended, could be achieved in 43 of 45
PDT-sessions receiving 100 J/cm2. The most prominent side effects were postoperative urgency and bladder pain, all
symptoms being more severe after 16 mM h-ALA. Preliminary evaluation shows complete response assessed at 3
months after the third PDT-session (i.e. 6 months after first treatment) in 9 of 12 patients. 2 of these patients were free of
recurrence until final follow-up at 84 weeks.
Prevention of bladder tumor implantion after fluorescence-guided TUR with photodynamic therapy
Author(s):
Saoussen Berrahmoune;
Lina Bezdetnaya;
Peter de Witte;
Agnès Leroux;
Dominique Dumas;
François Guillemin;
Marie Ange D'Hallewin
Show Abstract
The prevalence of bladder cancer is very high, due to its high recurrence rate in superficial bladder
cancer (30 to 85%), which is the staging of approximately 80% of the patients at first diagnosis. Risk of
recurrence and progression is associated with grade, stage, presence of concomitant carcinoma in situ, size and
number of lesions, as well as time to first recurrence. Recurrences can be partly attributed to new occurrences
but also to residual tumors after resection. Incomplete tumor removal has been observed in 30 to 50% of TUR's,
especially when dealing with T1 or poorly visible malignant or pre-malignant disease1. Fluorescence guided
resection with 5 amino levulinic acid (ALA) or its hexyl ester derivative (Hexvix, has now unequivocally
been demonstrated to increase detection rate and a growing number of studies indicate this has a positive impact
on recurrence and progression ratesImplantation of viable tumor cells, dispersed during resection, is a third factor influencing bladder cancer recurrence. The aim of early intravesical therapy is to interfere with cell
viability and thus reduce implantation risks.
A light blanket for intraoperative photodynamic therapy
Author(s):
Yida Hu;
Ken Wang;
Timothy C. Zhu
Show Abstract
A novel light source - light blanket composed of a series of parallel cylindrical diffusing fibers (CDF) is designed to
substitute the hand-held point source in the PDT treatment of the malignant pleural or intraperitoneal diseases. It
achieves more uniform light delivery and less operation time in operating room. The preliminary experiment was
performed for a 9cmx9cm light blanket composed of 8 9-cm CDFs. The linear diffusers were placed in parallel fingerlike
pockets. The blanket is filled with 0.2 % intralipid scattering medium to improve the uniformity of light
distribution. 0.3-mm aluminum foil is used to shield and reflect the light transmission. The full width of the profile of
light distribution at half maximum along the perpendicular direction is 7.9cm and 8.1cm with no intralipid and with
intralipid. The peak value of the light fluence rate profiles per input power is 11.7mW/cm2/W and 8.6mW/cm2/W
respectively. The distribution of light field is scanned using the isotropic detector and the motorized platform. The
average fluence rate per input power is 8.6 mW/cm2/W and the standard deviation is 1.6 mW/cm2/W for the scan in air,
7.4 mW/cm2/W and 1.1 mW/cm2/W for the scan with the intralipid layer. The average fluence rate per input power and
the standard deviation are 20.0 mW/cm2/W and 2.6 mW/cm2/W respectively in the tissue mimic phantom test. The light
blanket design produces a reasonably uniform field for effective light coverage and is flexible to confirm to anatomic
structures in intraoperative PDT. It also has great potential value for superficial PDT treatment in clinical application.
Preliminary study of verteporfin photodynamic therapy in a canine prostate model
Author(s):
Zheng Huang;
Fred Hetzel;
Ken Dole;
David Luck;
Jill Beckers;
Don Maul
Show Abstract
Photodynamic therapy (PDT) mediated with verteporfin was investigated as an
alternative modality for the treatment of prostate cancer. Materials and Methods: Vertoporfin-mediated
photodynamic effects on the prostate and its adjacent structures (underlying colon and
bladder) were evaluated in a healthy canine model. Interstitial prostate PDT was performed by
irradiating individual lobes with a diode laser (689 nm) and 1-cm cylindrical diffuser fibers at
various light doses and drug-light intervals (DLI) to activate the IV administrated photosensitizer
(0.5 or 2 mg/kg). The sensitivity of the adjacent tissues to Vertoporfin-PDT was determined by
superficially irradiating the serosal surface of the bladder and colon with a microlens fiber. The
prostate and adjacent tissues were harvested one-week after the treatment and subjected to
histopathological examination. Results: Histopathological examinations confirmed that verteporfin
PDT could destroy a clinically significant volume of prostatic tissue in the animal model. At the
drug dose of 0.5 mg/kg, the light irradiation of 100 J/cm could induce a lesion diameter of 2 cm at
DLI of 15 min and 1.2 cm at DLI of 3 hrs, respectively. This implies a strong influence of DLI on
the lesion volume. The shorter DLI might produce stronger vascular effect and therefore more
severe tissue damage. The colon was more sensitive to verteporfin PDT than the bladder. At the
possible light dose level caused by light scattering during intra-prostate irradiation, the damage to
the bladder and colon were superficial and minimal. Conclusions: The preliminary results clearly
demonstrate that verteporfin PDT could be an effective means to destroy prostate gland and its
usefulness for the treatment of prostate cancer is worth further investigation.
Topical application of ALA PDT for the treatment of moderate to severe acne vulgaris
Author(s):
Xiu-Li Wang;
Hong-Wei Wang;
Ling-Lin Zhang;
Lina Su;
Ming-Xia Guo;
Zheng Huang
Show Abstract
Objectives: To evaluate the effectiveness of topical 5-aminolevulinic acid (ALA)-
medicated photodynamic therapy (ALA PDT) for the treatment of moderate to severe acne
vulgaris. Methods: Sixteen Chinese patients with moderate to severe facial acne were
treated with 1-3 courses of ALA PDT. ALA cream (3%) was freshly prepared and applied
to acne lesions for 3-4 h. The lesions were irradiated by a 635 nm diode laser at dose
levels of 60 - 80 J/cm2 at 100 mW/cm2. Clinical assessments were conducted before and
after treatment up to 3 months. Results: All patents showed response to ALA PDT.
Complete clearance was seen in 10 patients (62.5%) and partial clearance in 6 patients
(37.5%). One case showed recurrence after complete clearance at 2 months and another
two showed recurrence after complete clearance at 3 months. However, the number of new
lesions were significantly reduced. Adverse effects were minimal. Conclusions: The
results of this preliminary clinical study is encouraging. ALA PDT is a simple, safe and
useful therapeutic option for the treatment of moderate to severe acne. Further studies to
evaluate the treatment with a larger number of patients and for a longer period of follow-up
are needed.
Using iron chelating agents to enhance dermatological PDT
Author(s):
Alison Curnow;
Yuktee Dogra;
Paul Winyard;
Sandra Campbell
Show Abstract
Topical protoporphyrin IX (PPIX) induced photodynamic therapy (PDT) of basal cell carcinoma (BCC) produces good
clinical outcomes with excellent cosmesis as long as the disease remains superficial. Efficacy for nodular BCC however
appears inferior to standard treatment unless repeat treatments are performed. Enhancement is therefore required and is
possible by employing iron chelating agents to temporarily increase PPIX accumulation above the levels normally
obtained using aminolevulinic acid (ALA) or the methyl ester of ALA (MAL) alone. In vitro studies investigated the
effect of the novel iron chelator, CP94 on necrotic or apoptotic cell death in cultured human skin fibroblasts and
epidermal carcinoma cells incubated with MAL. Furthermore, following a dose escalating safety study conducted with
ALA in patients, an additional twelve nodular BCCs were recruited for topical treatment with standard MAL-PDT +/-
increasing doses of CP94. Six weeks later following clinical assessment, the whole treatment site was excised for
histological analysis. CP94 produced greater cell death in vitro when administered in conjunction with MAL than this
porphyrin precursor could produce when administered alone. Clinically, PDT treatment using Metvix + CP94 was a
simple and safe modification associated with a trend of reduced tumor thickness with increasing CP94 dose.
Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation
Author(s):
Raghavan Rajagopalan;
Amol Karwa;
Przemyslaw M. Lusiak;
Kripa Srivastava;
Amruta R. Poreddy;
Raghootama S. Pandurangi;
Karen P. Galen;
William L. Neumann;
Gary E. Cantrell;
Richard B. Dorshow
Show Abstract
Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type
1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of
molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-)
functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared
and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell
binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at
various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers
caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly,
selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1
sensitizers are useful for phototherapeutic applications.
The correlation between photosensitizers' membrane localization, membrane-residing targets, and photosensitization efficiency
Author(s):
Shany Ytzhak;
Shoshana Bernstein;
Leslie M. Loew;
Benjamin Ehrenberg
Show Abstract
Various tetrapyrroles act as photosensitizers by efficiently generating singlet oxygen. Hydrophobic or amphiphilic
photosensitizers are taken up by cells and are usually located in various cellular lipid membranes. Passive uptake by a
membrane depends on biophysical properties of the membrane, such as its composition, temperature, phase, fluidity,
electric potential etc., as well as on the external solution's properties. Although the intrinsic lifetime of singlet oxygen in
the membrane phase is 10-30 μs, depending on lipid composition, it escapes much faster out of the membrane into the
external or internal aqueous medium, where its lifetime is <3 μs. Any damage that singlet oxygen might inflict to
membrane constituents, i.e. proteins or lipids, must thus occur while it is diffusing in the membrane. As a result,
photosensitization efficiency depends, among others, on the location of the sensitizer in the membrane. Singlet oxygen
can cause oxidative damage to two classes of targets in the membrane: lipids and proteins. Depolarization of the Nernst
electric potential on cells' membranes was observed, but it is not clear whether lipid oxidation is a relevant factor leading
to abolishing the resting potential of cells' membranes and to their death. We present a study of the effect of membrane
lipid composition and the dissipation of the electric potential that is generated across the membrane. We find a clear
correlation between the structure and unsaturation of lipids and the leakage of the membrane, which can be caused by
their photosensitized oxidization. We demonstrate here that when liposomes are composed of mixtures similar to natural
membranes, and photosensitization is being carried out under usual PDT conditions, photodamage to the lipids is not
likely to cause enhanced permeability of ions through the membrane, which could be a mechanism that leads to cell
death.
Introduction of an ultrathin and flexible laser scanning endoscope for color imaging and integrated PDD and future PDT
Author(s):
Eric J. Seibel;
Heidi L. Kenerson;
Cameron M. Lee;
C. David Melville;
Richard S. Johnston;
Raymond S. Yeung
Show Abstract
The scanning fiber endoscope (SFE), an ultrathin laser scanning endoscope capable of producing 500-line color
images at 30-Hz frame rate, has been developed at the University of Washington. The SFE probe is a 1-mm
diameter by 9-mm long rigid scanner at the tip of a highly flexible and robust tether (minimum bend radius < 8-
mm), comprised of helically wound optical fibers and electrical wires within a protective sheath. The unique
physical characteristics of this system have allowed the camera to navigate narrow passages where other
technologies have suffered from reduced image quality and fragility, such as imaging the peripheral airways and bile
duct. The scanning engine of the SFE allows for laser-based imaging and potential applications of pixel-accurate
therapy in remote regions of the body. In this study, the standard SFE operation has been tailored to create widefield
fluorescence images for photodynamic detection. A kidney with renal cell carcinoma was excised from an
Eker rat after post-mortem in situ perfusion with 0.4 mM hypericin. The 442-nm blue and 532-nm green laser
illumination sources were used for both standard reflectance imaging and fluorescence excitation, while the red 635-
nm illumination was disabled. Red detection signal gain was increased to amplify the red fluorescence signal from
the photomultiplier tube and within the computer image display. Results show green and blue reflectance images
overlaid with red fluorescence signal in tumor regions of the kidney. These imaging capabilities portend future
adoption of laser-based SFE imaging for real-time PDD.
Beta-lactamase targeted enzyme activatable photosensitizers for antimicrobial PDT
Author(s):
Xiang Zheng;
Sarika Verma;
Ulysses W. Sallum;
Tayyaba Hasan
Show Abstract
Photodynamic therapy (PDT) as a treatment modality for infectious disease has shown promise. However, most of the
antimicrobial photosensitizers (PS) non-preferentially accumulate in both bacteria and host tissues, causing host tissue
phototoxicity during treatment. We have developed a new antimicrobial PDT strategy which exploits beta-lactam
resistance mechanism, one of the major drug-resistance bacteria evolved, to achieve enhanced target specificity with
limited host damage. Our strategy comprises a prodrug construct with a PS and a quencher linked by beta-lactam ring,
resulting in a diminished phototoxicity. This construct, beta-lactamase enzyme-activated-photosensitizer (beta-LEAP),
can only be activated in the presence of both light and bacteria, and remains inactive elsewhere such as mammalian
tissue. Beta-LEAP construct had shown specific cleavage by purified beta-lactamase and by beta-lactamase over-expressing
methicillin resistant Staphylococcus aureus (MRSA). Specific photodynamic toxicity was observed towards
MRSA, while dark and light toxicity were equivalent to reference strains. The prodrug design, synthesis and
photophysical properties will be discussed.
New porphyrin glyco-conjugates
Author(s):
Charles Michael Drain;
Sunaina Singh;
Diana Samaroo;
Sebastian Thompson;
Mikki Vinodu;
Joao P. C. Tome
Show Abstract
Porphyrins bearing sugars and other motifs are proposed for a variety of therapeutic applications. Non-hydrolysable
glyco conjugates of porphyrins can be formed in rapid, room temperature reacting in greater than 90% yields from
tetraperfluorophenyporphyrin. Additional functional groups can be appended using the same chemistry but different
stoichiometries of the reagents. Thus sugars, amines, peptides, and cationic moieties designed to target cancer cells or
other diseased or disease-causing cells are made rapidly and cleanly. These compounds can then be rapidly screened for
cell uptake, or selected from combinatorial libraries by cell uptake assays using a combination of fluorescence
microscopy and mass spectrometry. Modifications of the macrocycle allow fine-tuning of the photonic properties for
specific medical, imaging, or biochemical applications.
Photodynamic synchrotron x-ray therapy in glioma cell using superparamagnetic iron nanoparticle
Author(s):
Hong-Tae Kim;
Ki-Hong Kim;
Gi-Hwan Choi;
Sanghoon Jheon;
Sung-Hwan Park;
Bong-Il Kim;
Kazuyuki Hyodo;
Masami Ando;
Jong-Ki Kim
Show Abstract
In order to evaluate cytotoxic effects of secondary Auger electron emission(Photon Activation Therapy:PAT)
from alginate-coated iron nanoparticles(Alg-SNP), Alg-SNP-uptaken C6 glioma cell lines were irradiated with 6.89/7.2
Kev synchrotron X-ray. 0-125 Gy were irradiated on three experimental groups including No-SNP group incubating
without SNP as control group, 6hr-SNP group incubating with SNP for 6hr and ON-SNP group incubating with SNP
overnight. Irradiated cells were stained with Acridine Orange(AO) and Edithium Bromide(EB) to count their viability
with fluorescent microscopy in comparison with control groups. AO stained in damaged DNA, giving FL color change in
X-ray plus SNP group. EB did not or less enter inside the cell nucleus of control group. In contrast, EB entered inside the
cell nucleus of Alg-SNP group which means more damage compared with Control groups. The results of MTT assay
demonstrated a X-ray dose-dependent reduction generally in cell viability in the experimental groups. 3 or 9 times
increase in cell survival loss rate was observed at 6hr-SNP and ON-SNP groups, respectively compared to No-SNP
control group in first experiment that was done to test cell survival rate at relatively lower dose, from 0 to 50 Gy. In
second experiment X-ray dose was increased to 125 Gy. Survival loss was sharply decreased in a relatively lower dose
from 5 to 25 Gy, and then demonstrated an exponentially decreasing behavior with a convergence until 125 Gy for each
group. This observation suggests PAT effects on the cell directly by X-ray in the presence of Alg-SNP occurs within
lower X-ray dose, and conventional X-ray radiation effect becomes dominant in higher X-ray dose. The cell viability
loss of ON-SNP group was three times higher compared with that of 6hr-SNP group. In conclusion, it is possible to
design photodynamic X-ray therapy study using a monochromatic x-ray energy and metal nanoparticle as x-ray sensitizer,
which may enable new X-ray PDT to disseminated tumors without side effects to normal surrounding tissue.
Clearance of protoporphyrin IX induced by 5-aminolevulinic acid from WiDr human colon carcinoma cells
Author(s):
Asta Juzeniene;
Miron Kaliszewski;
Andrzej Bugaj;
Johan Moan
Show Abstract
5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is the most widely practiced form of PDT in
dermatology. One of the advantages of ALA-PDT is that undesirable photosensitization lasts only for 24-48 h. In order
to optimize ALA-PDT it is necessary to understand the mechanisms controlling intracellular PpIX clearance (efflux and
transformation into heme) in order to decrease protoporphyrin IX (PpIX) clearance rates in the early stages of its
production. The aim of this study was to investigate the factors controlling the clearance of intracellular PpIX.
Fluorescence spectroscopy was used to study PpIX kinetics in WiDr cells initially treated with ALA. The clearance rate
of PpIX in WiDr cells was faster after application of a low concentration of ALA (0.1 mM) than after application of high
concentration of ALA (1 mM). PpIX was cleared faster from cells which initially were seeded at low densities than cells
seeded at higher densities. The presence of the iron chelator deferoxamine reduced the clearance rate of PpIX, while the
presence of ferrous sulfate acted oppositely. The decay rate of PpIX in WiDr cells was faster at higher temperature than
at lower. The ferrochelatase activity at pH 7.2 was significantly greater than that at pH 6.7. ALA concentration,
application time, cell density, temperature, pH, intracellular iron content, intracellular amount and localization of PpIX
are factors controlling PpIX clearance.
New stable synthetic bacteriochlorins for photodynamic therapy of melanoma
Author(s):
Pawel Mroz;
Ying-Ying Huang;
Sahar Janjua;
Timur Zhiyentayev;
Christian Ruzié;
K. Eszter Borbas;
Dazhong Fan;
Michael Krayer;
Thiagarajan Balasubramanian;
Eun Kyung Yang;
Hooi Ling Kee;
Dewey Holten;
Jonathan S. Lindsey;
Michael R. Hamblin
Show Abstract
Photodynamic therapy (PDT) has been successfully used to treat many malignancies, and has afforded highly
encouraging results in skin cancers such as basal cell carcinoma. However, pigmented melanoma remains a notable
exception from the range of tumors treated by PDT largely due to the fact that melanin has high absorption of light in
wavelength regions where most clinically approved photosensitizers (PS) absorb light (600-690 nm). Moreover,
melanoma cells sequester exogenous molecules including photosensitizers inside melanosomes. The aforementioned
drawbacks of the clinically used PS have motivated us to search for new classes of PS with improved spectral properties,
such as bacteriochlorins (BC) to be used in PDT of melanoma. To overcome the PDT-resistance mechanisms of
melanoma, particularly the high optical absorption of melanin, three near-infrared (NIR) absorbing synthetic stable BC
were used in PDT treatment of melanoma. Dose and fluence dependent cell killing, intracellular localization (particularly
in melanosomes), and correlation between the melanin level and cell death were examined. Intracellular melanosomes
are ruptured after illumination as shown by electron microscopy. The best in vitro performing BC were tested upon
delivery in micellar nanoparticles against a mouse pigmented melanoma. Two of the BC were effective at significantly
lower concentrations (<0.5 μM) than common photosensitizers in present use.
Selenium enhances the efficacy of Radachlorin-mediated photodynamic therapy in cervical cancer model
Author(s):
Dong Han Bae;
Lan Ying Wen;
Su Mi Bae;
Uk Kang;
Keun Hee Kim;
Sang Hoon Jheon;
Jeong Sang Lee;
Woong Shick Ahn
Show Abstract
Selenium, an essential trace element possessing anti-carcinogenic properties, can induce apoptosis in cancer cells. Our
goal was to investigate the enhanced anti-tumor effects of photodynamic therapy (PDT) plus selenium in TC-1 tumor
cells implanted into mice. The MTT assay and tumor growth inhibition study were evaluated at various time intervals
after the PDT plus a various dose of selenium. Following Radachlorin injections after 3 hr, the mice were then
administrated selenium (2ug/kg b.w.) and then, tumors were treated with external light treatment (300 J/cm2). The
selenium was administered daily for 20 days. PDT or selenium was found to be more compared to control groups.
Moreover, the PDT combined with selenium demonstrated a significant suppression of tumor growth in vitro and in vivo.
The tumor growth by the PDT combined with selenium was significantly reduced. These data suggest that selenium plus
PDT can induce a significant tumor suppression response compared with PDT alone. Also, it can be an effective
approach to induce anti-cancer therapy strategy.
Combination of PDT and topical angiogenic inhibitor for treatment of port wine stain (PWS) birthmarks: a novel approach
Author(s):
Kaihua Yuan;
Qiaobing Huang;
Zheng Huang
Show Abstract
Port wine stain (PWS) birthmarks are a congenital cutaneous vascular malformation involving
ecstatic post-capillary venules. Current standard treatment for PWS is the pulsed dye laser (PDL).
Vascular-targeted photodynamic therapy (PDT) has been used for the treatment of PWS in China
since the early 1990's. Both can achieve a certain degree of color blanching in various types of
PWS lesions. However, the majority of PWS lesions require multiple treatments. Some PWS
lesions can recur or become darker after successful treatment. Recently, it has been proposed that
this phenomenon might be initiated by neoangiogenesis that can be caused by treatment via wound
healing response. The combined use of photothermolysis and a topical application of an angiogenic
inhibitor such as Imiquimod and Rapamycin, were evaluated in several pilot studies. It is
well-known that PDT can induce various host immune responses VEGF overexpression. Recent
clinical data also show that improved clinical outcomes are obtained through the combination of
ocular PDT and anti-VEGF therapy. This article will discuss rationales and implications of using
such a combination modality and highlight recent progress based on our clinical experience and
published data.
A pilot study comparing the pain sensations during PpIX build-up and clearance phases
Author(s):
P. Mikolajewska;
A. Juzeniene;
V. Iani;
H. Sollund;
G. Norsang;
J. Moan
Show Abstract
It has been speculated that topical application of 5-aminolevulinic acid (ALA) or methyl 5-aminolevulinate (MAL) may
be more painful during light exposure after longer application times of the compounds than after shorter times, even
though the same levels of protoporphyrin IX (PpIX) is produced in both cases.
The aim of our study was to investigate pain induction in the build-up and clearance phases of PpIX in the skin of
healthy volunteers. 0.6 mmol/g of ALA (10% wt/wt) and MAL (11% wt/wt) creams were applied on the volunteers. The
creams were maintained on the spots for 20- 24 hours and then wiped off. Subsequently, fresh creams were applied on
the other arm of the volunteers for 4- 6 hours. Fluorescence emission spectra for all spots were measured every hour until
the fluorescence levels were similar in both arms for ALA and MAL. Then the test areas were exposed to light until pain
occurred. Time for pain to occur was recorded. The fluorescence of PpIX was measured before and after light exposure.
PDT in the clearance phase seems to induce pain faster than in the build-up phase for ALA and MAL. Due to large
interpersonal variations between volunteers further investigation is needed.
Results of combined photodynamic therapy (PDT) and high dose rate brachytherapy (HDR) in treatment of obstructive endobronchial non-small cell lung cancer
Author(s):
Benjamin D. Weinberg;
Ron R. Allison;
Claudio Sibata;
Teresa Parent;
Gordon Downie
Show Abstract
We reviewed the outcome of combined photodynamic therapy (PDT) and high dose rate brachytherapy
(HDR) for patients with symptomatic obstruction from endobronchial non-small cell lung cancer. Methods: Nine
patients who received combined PDT and HDR for endobronchial cancers were identified and their charts reviewed.
The patients were eight males and one female aged 52-73 at diagnosis, initially presenting with various stages of disease:
stage IA (N=1), stage IIA (N=1), stage III (N=6), and stage IV (N=1). Intervention was with HDR (500 cGy to 5 mm
once weekly for 3 weeks) and PDT (2 mg/kg Photofrin, followed by 200 J/cm2 illumination 48 hours post infusion).
Treatment group 1 (TG-1, N=7) received HDR first; Treatment group 2 (TG-2, N=2) received PDT first. Patients were
followed by regular bronchoscopies. Results: Treatments were well tolerated, all patients completed therapy, and none
were lost to follow-up. In TG-1, local tumor control was achieved in six of seven patients for: 3 months (until death), 15
months, 2+ years (until death), 2+ years (ongoing), and 5+ years (ongoing, N=2). In TG-2, local control was achieved in
only one patient, for 84 days. Morbidities included: stenosis and/or other reversible benign local tissue reactions (N=8);
photosensitivity reaction (N=2), and self-limited pleural effusion (N=2). Conclusions: Combined HDR/PDT treatment
for endobronchial tumors is well tolerated and can achieve prolonged local control with acceptable morbidity when PDT
follows HDR and when the spacing between treatments is one month or less. This treatment regimen should be studied
in a larger patient population.
Photodynamic therapy of cervical intraepithelial neoplasia using hexaminolevulinate and methylaminolevulinate
Author(s):
Philipp Soergel;
Ismini Staboulidou;
Herrmann Hertel;
Cordula Schippert;
Peter Hillemanns
Show Abstract
Cervical intraepithelial neoplasia (CIN) is the precursor of invasive cervical cancer. Previous studies indicated that
photodynamic therapy (PDT) represents an effective treatment modality in CIN. In 28 patients with CIN 1 - 3, 1 - 2
cycles of PDT were conducted using hexaminolevulinate (HAL) or methylaminolevulinate (MAL) and a special light
delivery system. After 6 months, biopsies were obtained to assess response. The overall response rate for complete or
partial response was 65%. Photodynamic therapy using new ALA esters is effective and may offer unique advantages in
the therapy of CIN.
Combination of vascular targeting PDT with combretastatin A4 phosphate
Author(s):
Chong He;
Babasola Fateye;
Bin Chen
Show Abstract
Tumor vasculature is an attractive target for cancer therapy due to its accessibility to blood-borne therapeutic agents and
the dependence of tumor cells on a functional blood supply for survival and growth. Vascular targeting photodynamic
therapy (vPDT) is a novel modality based on the selective laser light activation of photosensitizers localized inside tumor
vasculature to shutdown tumor vascular function. Although this vascular targeting therapy is showing great promise for
cancer treatment, tumor recurrence has been observed in both preclinical and clinical studies. In this study, we intend to
enhance the therapeutic outcome of vascular targeting PDT by combining it with combretastatin A4 phosphate (CA4P), a
blood flow inhibitor. We found that the combination of CA4P and vPDT significantly increased endothelial cell
apoptosis than each single therapy. Western blot analysis suggests that myosin light chain kinase (MLCK) is a common
target of CA4P and vPDT. In a PC-3 prostate tumor model, we found that CA4P was able to greatly enhance tumor
response to vPDT. These results demonstrate that CA4P and vPDT can be combined to enhance the therapeutic effect.
HAL fluorescence cystoscopy: towards a new paradigm in bladder cancer management
Author(s):
Patrice Jichlinski
Show Abstract
Hexaminolevulinate fluorescence cystoscopy by the addition of a specific tissue fluorophore enhances the
contrast between benign and malignant epithelial (urothelial) cells in the bladder. Improved detection of flat and
papillary tumors as confirmed in recent phase III clinical trials allows the urologist to obtain a precise
information on the disease extent at the whole surface of the bladder wall. With gaining experience, management
of non muscle invasive bladder cancer improves in readiness and accuracy.
Antimicrobial photodynamic therapy in a mouse model of Acinetobacter baumannii burn infection
Author(s):
Tianhong Dai;
George P. Tegos;
Zongshun Lu;
Timur Zhiyentayev;
Liyi Huang;
Michael J. Franklin;
David G Baer;
Michael R. Hamblin
Show Abstract
Multi-drug resistant Acinetobacter baumanii infections represent a growing problem, especially in traumatic wounds and
burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment using traditional antibiotics
can be extremely difficult and new antimicrobial approaches are being investigated. One of these antimicrobial
alternatives could be the combination of non-toxic photosensitizers (PS) and visible light known as photodynamic
therapy (PDT). We report on the establishment of a new mouse model of full thickness thermal burns infected with a
bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS
produced by covalent conjugation chlorin(e6) to polyethylenimine followed by illumination of the burn surface with red
light. Application of 108 A. baumannii cells to the surface of 10-second burns made on the dorsal surface of shaved
female BALB/c mice led to chronic infections that lasted on average 22 days characterized by a remarkably stable
bacterial bioluminescence. PDT carried out on day 0 soon after applying bacteria gave over three logs of loss of bacterial
luminescence in a light exposure dependent manner, while PDT carried out on day 1 and day 2 gave approximately a
1.7-log reduction. Application of PS dissolved in 10% or 20% DMSO without light gave only modest reduction in
bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally
modest. It was also found that PDT did not lead to inhibition of wound healing. The data suggest that PDT may be an
effective new treatment for multi-drug resistant localized A. baumannii infections.
Investigation into the susceptibility of Burkholderia cepacia complex isolates to photodynamic antimicrobial chemotherapy (PACT)
Author(s):
C. M. Cassidy;
A. L. Watters;
R. F. Donnelly;
M. M. Tunney
Show Abstract
The main cause of morbidity and mortality in cystic fibrosis (CF) sufferers is progressive pulmonary damage caused by
recurrent and often unremitting respiratory tract infection. Causative organisms include Pseudomonas aeruginosa and
Haemophilus influenzae, but in recent years the Burkholderia cepacia complex has come to the fore. This group of
highly drug-resistant Gram-negative bacteria are associated with a rapid decline in lung function and the often fatal
cepacia syndrome, with treatment limited to patient segregation and marginally effective antibacterial regimens. Thus,
development of an effective treatment is of the upmost importance. PACT, a non-target specific therapy, has proven
successful in killing both Gram-positive and Gram-negative bacteria. In this study, planktonic cultures of six strains of
the B. cepacia complex were irradiated (635 nm, 200 J cm-2,10 minutes irradiation) following 30 seconds incubation
with methylene blue (MB) or meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Rates of kill of > 99 %
were achieved with MB- and TMP-PACT. A MB concentration of 50 μg ml-1 and TMP concentration of 500 μg ml-1
were associated with highest percentage kills for each photosensitizer. PACT is an attractive option for treatment of
B.cepacia complex infection. Further study, involving biofilm culture susceptibility, delivery of light to the target and
in vivo testing will be necessary before it PACT becomes a viable treatment option for CF patients who are colonised or
infected with B. cepacia complex.
Antimicrobial photodynamic therapy for the decolonization of methicillin-resistant Staphylococcus aureus from the anterior nares
Author(s):
Cale N. Street;
Lisa Pedigo;
Aaron Gibbs;
Nicolas G. Loebel
Show Abstract
The nosocomial infection rate has increased dramatically due to emergence of antibiotic resistant bacterial strains such as
methicillin resistant Staphylococcus aureus (MRSA). The primary anatomical site of MRSA colonization is the anterior
nares, and this reservoir represents a primary vector of transmission from non-infected carriers to susceptible individuals.
Antimicrobial photodynamic therapy (aPDT) has been used successfully for topical disinfection in the oral cavity. The
aim of this study was to evaluate the utility of aPDT for nasal MRSA decolonization at the preclinical and clinical level.
The nasal aPDT system consists of a 670 nm diode laser fibre-optically coupled to a disposable light diffusing tip, used
to activate a methylene blue based photosensitizer formulation. Preclinical testing was done both in a custom nasal
reservoir model and on human skin cultures colonized on the epithelial surface with MRSA. Human clinical testing was
performed by clinicians in regions in which the system is approved by the regulatory authority. In vitro testing
demonstrated that aPDT eradicated planktonic MRSA in an energy and photosensitizer concentration dependent manner.
Furthermore, aPDT eliminated sustained colonization of MRSA on cultured human epithelial surfaces, an effect that was
sustained over multiple days post-treatment. In preliminary human testing, aPDT eradicated MRSA completely from the
nose with total treatment times <10 minutes. aPDT is effective against MRSA when used topically in the nose. Energy
dose and photosensitizer parameters have been optimized for the nasal environment. Controlled clinical studies are
currently underway to further evaluate safety and efficacy.
Influence of bacterial interactions on the susceptibility to photodynamic inactivation
Author(s):
M. H. Upadya;
G. Tegos;
M. Hamblin;
A. Kishen
Show Abstract
Photodynamic therapy has emerged as a possible supplement to the existing protocols for endodontic
disinfection. Microbes are known to gain significant ecological advantage when they survive as
coaggregates and biofilms in an infected tissue. Such microbial coaggregates and biofilms have been
confirmed to play a key role in the pathogenicity of many infections. So far, not many studies have
correlated the efficacy of antimicrobial photodynamic inactivation (APDI) to the different modes of
bacterial growth. This study aims to evaluate the APDI of 3 strains of Enterococcus faecalis in planktonic
phase, in a co-aggregated suspension and in a 4-day old biofilm. The results showed that the biofilm mode
of growth offered the greatest resistance to APDI and the inclusion of an efflux pump inhibitor significantly
increased the APDI of biofilm bacteria. From this study, we conclude that APDI of bacteria in biofilms is
the most challenging and that the use of bacterial efflux pump inhibitors enhances its photodynamic antibiofilm
efficacy.
Photodynamic dosimetry in the treatment of periodontitis
Author(s):
Roger C. Andersen;
Nicolas G. Loebel;
Dane M. Andersen
Show Abstract
Photodynamic therapy has been demonstrated to effectively kill human periopathogens in vitro. However, the translation
of in vitro work to in vivo clinical efficacy has been difficult due to the number of variables present in any given patient.
Parameters such as photosensitizer concentration, duration of light therapy and amount of light delivered to the target
tissue all play a role in the dose response of PDT in vivo. In this 121 patient study we kept all parameters the same except
for light dose which was delivered at either 150 mW or 220 mW. This clearly demonstrated the clinical benefits of a
higher light dose in the treatment of periodontitis.
Photodynamic inactivation of antibiotic resistant strain of Pseudomonas aeruginosa in vivo
Author(s):
M. C. E. Hashimoto;
D. J. Toffoli;
R. A. Prates;
Lilia Coronato Courrol;
M. S. Ribeiro
Show Abstract
Burns are frequently contamined by pathogenic microorganisms and the widespread occurrence of antibiotic resistant
strains of Pseudomonas aeruginosa in hospitals is a matter of growing concern. Hypocrellin B (HB) is a new generation
photosensitizer extracted from the fungus Hypocrella bambusae with absorption bands at 460, 546 and 584 nm.
Lanthanide ions change the HB molecular structure and a red shift in the absorption band is observed as well as an
increase in the singlet oxygen quantum yield. In this study, we report the use of HB:La+3 to kill resistant strain of P.
aeruginosa infected burns. Burns were produced on the back of mice and wounds were infected subcutaneously with
1x109 cfu/mL of P. aeruginosa. Three-hours after inoculation, the animals were divided into 4 groups: control, HB:La+3,
blue LED and HB:La+3+blue LED. PDT was performed using 10μM HB:La+3 and 500mW light-emitting diode (LED)
emitting at λ=470nm±20nm during 120s. The animals of all groups were killed and the infected skin was removed for
bacterial counting. Mice with photosensitizer alone, light alone or untreated infected wounds presented 1x108 cfu/g while
mice PDT-treated showed a reduction of 2 logs compared to untreated control. These results suggest that HB:La+3
associated to blue LED is effective in diminishing antibiotic resistant strain P. aeruginosa in infected burns.
Photochemistry-based immune modulation in the treatment of cutaneous leishmaniasis
Author(s):
Oleg E Akilov;
Sachiko Kosaka;
Tayyaba Hasan
Show Abstract
The destruction of infectious pathogens by photodynamic therapy (PDT) is an emerging modality.
We demonstrated the efficacy of PDT for the management of cutaneous leishmaniasis in our
previous studies. However, much remains to be done for the improvement of PDT regimens. The
modulation of the immune response by photochemistry is an exciting but under-explored area of
PDT research. The goal of this study is to understand the mechanisms of the augmentation of the
host immune response after PDT of cutaneous leishmaniasis (CL). We found that PDT with
phenoxiazine analogues was capable for induction of Th1 immune response due to stimulation of IL-
12 production by dendritic cells. Single PDT treatment facilitated fast healing of the CL lesions due
to effective parasite eradication and augmentation of the immune system. Comparative study with
different photosensitizers (PS) (porphyrins, pehnoxiazines) demonstrated different
immunomodulating properties of PDT depending on chemical class of PS. Knowing the particular
profiles and immunomodulating properties of the pertinent PSs allows us to select the optimal PS
with regards to both the photodestructive and immunostimulating potential.
Absence of bacterial resistance following repeat exposure to photodynamic therapy
Author(s):
Lisa A. Pedigo;
Aaron J. Gibbs;
Robert J. Scott;
Cale N. Street
Show Abstract
The prevalence of antibiotic resistant bacteria necessitates exploration of alternative approaches to treat hospital and
community acquired infections. The aim of this study was to determine whether bacterial pathogens develop resistance
to antimicrobial photodynamic therapy (aPDT) during repeated sub-lethal challenge. Antibiotic sensitive and resistant
strains of S. aureus and antibiotic sensitive E. coli were subjected to repeat PDT treatments using a methylene blue
photosensitizer formulation and 670 nm illumination from a non-thermal diode laser. Parameters were adjusted such that
kills were <100% so that surviving colonies could be passaged for subsequent exposures. With each repeat, kills were
compared to those using non-exposed cultures of the same strain. Oxacillin resistance was induced in S. aureus using a
disc diffusion method. For each experiment, "virgin" and "repeat" cultures were exposed to methylene blue at 0.01%
w/v and illuminated with an energy dose of 20.6 J/cm2. No significant difference in killing of E. coli (repeat vs. virgin
culture) was observed through 11 repeat exposures. Similar results were seen using MSSA and MRSA, wherein kill rate
did not significantly differ from control over 25 repeat exposures. In contrast, complete oxacillin resistance could be
generated in S. aureus over a limited number of exposures. PDT is effective in the eradication of pathogens including
antibiotic resistance strains. Furthermore, repeated sub-lethal exposure does not induce resistance to subsequent PDT
treatments. The absence of resistance formation represents a significant advantage of PDT over traditional antibiotics.
Photodynamic inactivation of Gram (-) and Gram (+) microorganisms by cationic porphyrins and metalloporphyrins
Author(s):
Grigor V. Gyulkhandanyan;
Marina H. Paronyan;
Anichka S. Hovsepyan;
Robert K. Ghazaryan;
Artak G. Tovmasyan;
Aram G. Gyulkhandanyan;
Anna G. Gyulkhandanyan;
Gayane V. Amelyan
Show Abstract
Photodynamic inactivation (PDI) of microorganisms is successfully applied against Gram (+)
microorganisms. However the majority of photosensitizers poorly affect on Gram (-) microorganisms. At
present number of works have shown that cationic photosensitizers are able to induce photoinactivation both
Gram (+) and Gram (-) microorganisms. The purpose of this work was definition of more effective new
cationic pyridylporphyrins with various coordinated metals and functional groups for destruction of Gram (-)
microorganisms. The efficiency of new cationic porphyrins and metalloporphyrins (9 compounds) was tested
against Gram (-) microorganism E. coli (strain Κ-12). The testing results show high efficiency of
metalloporphyrins, especially silver complexes, against E. coli microorganism under dark conditions. 50 %
and 100 % cell growth inhibitory concentrations (IC50 and IC100 values, accordingly) of studied metallocomplexes
are considerably lower in comparison with metal-free porphyrins. At the same time the Zncomplexes
of porphyrins are more phototoxic than their metal-free analogues. Zn-metalloporphyrins with
allyl and butyl functional groups were shown the highest efficiency against E. coli. The photodynamic action
of cationic Zn -TBut4PyP metalloporphyrin against Gram(+) (St. aureus and St. epidermis) and Gram(-)
(E.coli, strain K-12 and Salmonella sp.) microorganisms was investigated. It is revealed, that Gram (+)
microorganisms were 3-5 times more susceptible to the compounds' phototoxic influence than Gram (-)
microorganisms.
Strategies for targeted antimicrobial photodynamic therapy
Author(s):
Sarika Verma;
Ulysses Sallum;
Xiang Zheng;
Tayyaba Hasan
Show Abstract
The photophysics and mechanisms of cell killing by photodynamic therapy (PDT) have been extensively studied in
recent years, and PDT has received regulatory approval for the treatment of a number of diseases worldwide. As the
application of this treatment modality expands with regard to both anatomical sites and diseases, it is important to
develop strategies for enhancing PDT outcomes. Our group has focused on developing targeting strategies to enhance
PDT for both cancerous as well as anti-microbial applications. In this article, we will discuss photosensitizer
modification and conjugation strategies for targeted antimicrobial photodynamic therapy.
A study of MRI-guided diffuse fluorescence molecular tomography for monitoring PDT effects in pancreas cancer
Author(s):
Kimberley S. Samkoe;
Scott C. Davis;
Subhadra Srinivasan;
Julia A. O'Hara;
Tayyaba Hasan;
Brian W. Pogue
Show Abstract
Over the last several decades little progress has been made in the therapy and treatment monitoring of pancreas
adenocarcinoma, a devastating and aggressive form of cancer that has a 5-year patient survival rate of 3%. Currently,
investigations for the use of interstitial Verteporfin photodynamic therapy (PDT) are being undertaken in both orthotopic
xenograft mouse models and in human clinical trials. In the mouse models, magnetic resonance (MR) imaging has been
used as a measure of surrogate response to Verteporfin PDT; however, MR imaging alone lacks the molecular
information required to assess the metabolic function and growth rates of the tumor immediately after treatment. We
propose the implementation of MR-guided fluorescence tomography in conjunction with a fluorescently labeled (IR-Dye
800 CW, LI-COR) epidermal growth factor (EGF) as a molecular measure of surrogate response. To demonstrate the
effectiveness of MR-guided diffuse fluorescence tomography for molecular imaging, we have used the AsPC-1
(+EGFR) human pancreatic adenocarcinoma in an orthotopic mouse model. EGF IRDye 800CW was injected 48 hours
prior to imaging. MR image sequences were collected simultaneously with the fluorescence data using a MR-coupled
diffuse optical tomography system. Image reconstruction was performed multiple times with varying abdominal organ
segmentation in order to obtain a optimal tomographic image. It is shown that diffuse fluorescence tomography of the
orthotopic pancreas model is feasible, with consideration of confounding fluorescence signals from the multiple organs
and tissues surrounding the pancreas. MR-guided diffuse fluorescence tomography will be used to monitor EGF
response after photodynamic therapy. Additionally, it provide the opportunity to individualize subsequent therapies
based on response to PDT as well as to evaluate the success of combination therapies, such as PDT with chemotherapy,
antibody therapy or even radiation.
Photofrin-PDT for gastric cancer in the era of endoscopic submucosal dissection
Author(s):
Yoshiro Nishiwaki;
Yoshito Ikematsu;
Yuuji Tokunaga;
Toshikazu Kanai
Show Abstract
Background: Endoscopic mucosal resection (EMR) was originated to treat early gastric cancer (EGC). EMR was suitable
for small, mucosal and well-differentiated adenocarcinoma without ulceration. It was difficult to resect larger tumors en
bloc by this method. In recent years, a more useful method, endoscopic submuscosal dissection (ESD) has been
developed, which enables en bloc resection of large mucosal lesions. On the contrary, photodynamic therapy (PDT) is
applicable to submucosal, poorly differentiated, or carcinoma with ulceration. In the era of ESD, we evaluated the value
of Photofrin-PDT. Patients & Methods: We applied PDT to 36 patients including three advanced cancers, who had been
excluded from EMR (ESD) and were at high risks for surgery or refused surgery. Four EGC patients who had not been
cured by EMR (ESD) were included. Our PDT procedure consisted of polyhematoporphyrin ether/ester administration
(Photofrin, 2 mg/Kg) and pulsed excimer dye laser irradiation at 630 nm 48 hours (and 96 hours) after sensitization.
Results: Complete response (CR) at three months was obtained in 84% (21/25) of mucosal cancer and in 50% (4/8) of
submucosal cancer. Although three patients with an advanced cancer improved but were not cured, quality of their life
was maintained. There were no serious side effects except skin photosensitivity. Conclusion: Photofrin-PDT should be
applied not only EGC patients who are excluded from ESD and have not been cured by ESD with poor risk for surgery,
and have high possibilitiy to be cured by PDT, but also advanced cancer patients for local improvement of lesions.
Correction of fluorescence for depth-specific optical and vascular properties using reflectance and differential path-length spectroscopy during PDT
Author(s):
F. van Zaane;
T. A. Middelburg;
H. S. de Bruijn;
A. van der Ploeg-van den Heuvel;
E. R. M. de Haas;
H. J. C. M. Sterenborg;
H. A. M. Neumann;
D. J. Robinson
Show Abstract
Introduction: The rate of PpIX fluorescence photobleaching is routinely used as a dose metric for ALA-PDT. Diffuse
reflection spectroscopy is often used to account for variations in tissue optical properties at the photosensitizer excitation
and emission bands. It can be used to quantify changes in vascular parameters, such as blood volume fraction and
saturation, and can aid understanding of tissue response to PDT. The volume and(/or) depth over which these signals are
acquired are critical. The aim of this study is to use quantitative reflectance spectroscopy (DPS) to correct fluorescence
for changes in tissue optical properties and monitor PDT.
Materials & Methods: ALA was topically applied to hairless mice skin and the incubated spot was treated with PDT
according to fractionated illumination schemes. DPS measurements of vascular parameters and optical properties were
performed directly before and after illumination. Both the differential signal, delivery-and-collection-fiber signal and the
collection fiber signal, which all probe different measurement volumes, are analyzed.
Results & Conclusions: Analysis of DPS measurements shows that at the depth where most fluorescence originates,
there is almost no blood present. During PDT vascular parameters at this depth stay constant. In more oxygenated layers
of the tissue, the optical properties do change during PDT, suggesting that only a small part of PpIX fluorescence
originates from the interesting depths where vascular response occurs. Correcting fluorescence emission spectra for
optical changes at specific depths and not for the total of changes in a larger volume, as is usually done now, can
improve PpIX photobleaching based treatment monitoring.
Islands of surviving cells within necrotic volume at liver induced by PDT
Author(s):
J. Ferreira;
C. Kurachi;
S. Zucoloto;
O. Castro e Silva Jr.;
V. S. Bagnato
Show Abstract
Tissue heterogeneities as well as distinct metabolic status and cellular types within a tumor may results in a nonhomogenous
necrosis. The possibility of PDT surviving cells within a treated volume has relevant clinical significance.
The major aim of this study was to analyze, on normal rat liver, the cell viability and surviving after PDT. The porphyrin
was injected through the cava vein at concentration of 1.5 mg/Kg. The induced necrosis was qualitatively investigated
varying the used drug light interval (30 min, 1, 3, 6, 24 and 36 h) and total delivered dose (20, 50, 100, 150 and 200
J/cm2). A diode laser at 630 nm and irradiance of 150 mW/cm2 was employed. The exposed livers were removed after
the animals were killed by anesthesia overdose, 30 h after illumination. Slides were processed by HE analysis for the
determination of the overall aspects of the necrotic and non-treated liver. We observed necrosis of central vein and
presence of surviving cell around the portal triad within the necrotic volume, suggesting PDT-resistant regions of the
tissue. Possible hypothesis for the observation may be: the absence of photosensitizer; insufficient light dose (below
threshold); and distinct metabolic status in the portal triad microregions decreases oxygen availability to photodynamic
reaction. The cells surrounding portal triad presented a higher resistance even when 200 J/cm2 was applied. In contrast,
the cells closer to the central vein after 20 J/cm2 were already susceptible to the action of PDT. Different aspects of the
problem are presented.
Vascular effects induced by anti-VEGF agents in the CAM model: effect of the DMSO
Author(s):
Patrycja Nowak-Sliwinska;
Jean-Pierre Ballini;
Hubert van den Bergh;
Georges Wagnières
Show Abstract
The chicken embryo's chorioallantoic membrane (CAM) is widely used as an in vivo model to study the vascular effects
induced by agents administrated topically or intravenously. Hence, in the vascular plexus of this respiratory membrane,
angiogenic and anti-angiogenic agents, as well as phototoxic effects have been studied.
The main goal of this study was to characterize the capillary network of the CAM after topical administration of
dimethyl sulfoxid (DMSO), a frequently used solvent of lipophylic drugs, including potent anti-VEGF agents. The CAM
capillaries were observed between days 8 and 9 of the embryo development, with an epi-fluorescence microscope
equipped with a sensitive camera by intravenous injection of a fluorescent agent and a non-fluorescing absorber (in the
extra-embryonic cavity) to screen the tissue background fluorescence. The fluorescence images of the CAM vasculature
were then processed in order to obtain a skeleton of the vessels and capillaries. This was done to quantify descriptors
such as the number of branching points/mm2, the mean area value of the vessels network meshes, and the mean of the 3rd
quartile of the histogram of these meshes, were then extracted.
Our results demonstrate that the topical administration of an aqueous solution of 20 μl of DMSO at concentrations equal
or larger than 0.1% turned out to modify the capillary network morphology in a dose-dependent manner as compared to
the control (20 μl of 0.9% NaCl).
PDT-induced in vitro bystander effect
Author(s):
David Olivier;
Samuel Douilard;
Thierry Patrice
Show Abstract
The mechanisms of Photodynamic therapy (PDT) include singlet oxygen and reactive oxygen species (ROS) production
that damage tumor cells and vasculature. The resulting effect is a balance between photo-oxidations via primary or
secondary ROS and scavenging activity. Sensitizers distribute in the extra-cellular space before and during cell
sensitization, suggesting that PDT could act directly on cell structures and on extra-cellular compartments, including
sera. In this paper we endeavored to determine whether the application of PDT to culture media could have an effect on
cell survival. Culture media (RPMI supplemented with Fetal Calf Serum (FCS)) was incubated with Rose Bengal (RB)
and irradiated before being added to cells for various times of contact, as a replacement for untreated media. Treatedmedia
reduced cell survival by up to 40% after 30 min of contact for 10 μg/mL of RB and 20 J/cm2. This effect was RB
or light dose-dependent. The survival reduction observed when adding treated-media was more pronounced when cell
doubling time was shorter. Analysis of ROS or peroxide production in treated-media revealed a long-lasting oxidizing
activity. Our findings support the hypothesis of a ROS or peroxide-mediated, PDT-induced, delayed cell toxicity
Targeted two-photon PDT photo-sensitizers for the treatment of subcutaneous tumors
Author(s):
C. W. Spangler;
A. Rebane;
J. Starkey;
M. Drobizhev
Show Abstract
New porphyrin-based photo-sensitizers have been designed, synthesized and characterized that exhibit greatly enhanced
intrinsic two-photon absorption. These new photo-sensitizers have been incorporated into triad formulations that also
incorporate Near-infrared (NIR) imaging agents, and small-molecule targeting agents that direct the triads to cancerous
tumors' over-expressed receptor sites. PDT can be initiated deep into the tissue transparency window at 780-800 nm
utilizing a regeneratively amplified Ti:sapphire laser using 100-150 fs pulses of 600-800 mW. Human tumor xenografts
of human breast cancer (MDA-MB-231) and both small SCLC (NCI-H69) and NSCLC (A-459) have been successfully
treated using octreotate targeting of over-expressed SST2 receptors. In particular, the lung cancer xenografts can be
successfully treated by irradiating from the side of the mouse opposite the implanted tumor, thereby passing through ca.
2 cm of mouse skin, tissue and organs with no discernible damage to healthy tissue while causing regression in the
tumors. These results suggest a new PDT paradigm for the noninvasive treatment of subcutaneous tumors, including the
possibility that the targeting moiety could be matched to individual patient genetic profiles (patient-specific
therapeutics).
Evaluation of toxicological properties and photodynamic activity of Photolon ointment: an experimental study
Author(s):
Siarhei V. Shliakhtsin;
Tatsiana V. Trukhachova;
Yuriy P. Istomin;
Ludmila N. Dunetz;
Andrey V. Kuvshinov;
Semen A. Naumovich
Show Abstract
The purpose of the present study was to evaluate toxicological properties and photodynamic activity of a new ready form
of the photosensitizer Photolon (Fotolon) - an ointment for topical use. The data obtained show the use of topicaly
applied photosensitizer provides sufficient penetration and accumulation of the active compound in tumor tissue as well
as in affected periodontal tissues for the effective PDT. There are several advantages of PDT with topical application of
the photosensitizer such as absence of systemic toxic and photosensitive reactions, relatively low cost of the treatment
and etc. We have shown that PDT of affected periodontal tissues with local application of Photolon/Fotolon ointment
provides an ability of local destruction of microbial cell, located as on the gum surface as in the spatium intercellulare
what is extremely important for successful treatment of acute and chronic periodontitis.
Anti-tumor effects on the combination of photodynamic therapy with arsenic compound in TC-1 cells implanted C57BL/6 mice
Author(s):
Kyu Wan Lee;
Lan Ying Wen;
Su Mi Bae;
Choong Hak Park;
Woo Kyu Jeon;
Doo Yun Lee;
Woong Shick Ahn
Show Abstract
The effects of As4O6 were studied as adjuvant on photodynamic therapy. As4O6 is considered to have anticancer activity
via several biological actions such as free radical producing and inhibition of VEGF expression. In vitro experiments,
cell proliferation and morphology were determined by MTT assay. Also, quantitative PCR array was performed to study
the synergetic mechanism. Additionally, this study was supported by the finding that combination of photodynamic
therapy and As4O6 shows an inhibition effect of tumor growth in C57BL/6 mice with TC-1 cells xenographs in vivo.
Radachlorin and As4O6 significantly inhibited TC-1 cell proliferation in a dose-dependent manner (P < 0.05). Antiproliferative
effect of combination treatment was significantly higher than those of TC-1 cells treated with either
photodynamic therapy or As4O6 (62.4 and 52.5% decrease, respectively, compared to photodynamic therapy or As4O6
alone, P < 0.05). In addition, cell proliferation in combination of photodynamic therapy and As4O6 treatment
significantly decreased by 77.4% compared to vehicle-only treated TC-1 cells (P < 0.05). Cell survival pathway (Naip1,
Tert and Aip1) and p53-dependent pathway (Bax, p21Cip1, Fas, Gadd45, IGFBP-3 and Mdm-2) were markedly increased
by combination treatment of photodynamic therapy and As4O6. Besides, the immunology response NEAT pathway (Ly-
12, CD178 and IL-2) also modulated after combination treatment of photodynamic therapy and As4O6. This combination
effect apparently shows a same pattern in vivo model. These findings suggest the benefit of the combination treatment of
photodynamic therapy and As4O6 for the inhibition of cervical cancer growth.
Endoscopic treatment of early bronchial cancer: our experience with photodynamic therapy (PDT)
Author(s):
Luigi Corti;
Lamberto Toniolo;
Caterina Boso;
Flavio Colaut;
Davide Fiore;
Pier-Carlo Muzzio;
Lucio Loreggian;
Guido Sotti
Show Abstract
The role of photodynamic therapy (PDT) in the treatment of small cancers has been
established in several clinical studies. Here, we report on the efficacy of PDT for
early inoperable or recurrent non-small-cell lung cancer (NSCLC). Methods and Materials: From
June 1989 to November 2004, 40 patients with 50 NSCLC were treated with PDT. Twelve cases
were inoperable for medical reasons and were staged as T1N0M0, and 28 had recurrent in situ
carcinoma. Patients with residual disease after PDT received definitive radiotherapy and/or
brachytherapy. Follow-up ranged from 6 to 167 months (median 43.59). Twenty of the 40 patients
received i.v. injections of hematoporphyrin derivative (5 mg/kg), the other 20 had injections of
porfimer sodium (Photofrin, 2 mg/kg). An argon dye laser (630 nm wavelength, 200-300 J/cm2) was
used for light irradiation in 24 of the 40 patients, a diode laser (Diomed, 630 nm wavelength, 100-
200 J/cm2) in the other 16. Results: PDT obtained a 72% complete response (CR) rate (36/50 treated
lesions), that is 27 CR among the 37 Tis carcinomas and 9 among the 13 T1 cases. Kaplan-Meier
curves showed a mean overall survival (OS) of 75.59 months (median 91.4 months). Two- and 5-
year OS rates were 72.78% and 59.55%. The mean and median survival rates for patients with Tis
stage were 86.5 and 120.4 months, respectively (standard error 9.50) and for patients with T1 disease
they were 45.78 and 35.71 months, respectively; the difference was statistically significant
(P< 0.03). No severe early or late PDT-related adverse events were recorded.
Conclusions: PDT is effective in early primary or recurrent NSCLC, resulting in a CR rate of 72%.
The incorporation of PDT in standard clinical practice, in combination with radiotherapy, warrants
further investigation.
Reconstruction of hemodynamics and sensitizer distributions during interstitial PDT using spectroscopy with linear light sources
Author(s):
Jarod C. Finlay;
Ken Wang;
Yida Hu;
Timothy C. Zhu
Show Abstract
Light dosimetry for photodynamic therapy requires a knowledge of the optical absorption spectrum of the tissue being
treated Here, we present a theoretical and experimental analysis of the capabilities of a system using interstitial linear
light sources ranging in length from 2 to 5 cm to illuminate the tissue interstitially, and isotropic point-like detectors to
measure the resulting diffusely transmitted light. The sources and detectors are translated in transparent plastic catheters
under the control of a motorized positioning system designed for interstitial measurements in the prostate. The light
source is a quartz-tungsten-halogen (QTH), and the spectrally resolved detection is accomplished using a CCD-based
grating spectrometer. The data are analyzed using an approximation to the radiative transport equation, assuming
homogeneous scattering and heterogeneous absorption spectra Absorption spectra are reconstructed independently for
individual source-detector channel pairs. Sequential reconstruction can then be used to create a 3-dimensional
reconstruction. The results of simulated data, measurements made in multi-component phantoms, and synthetic data
reconstructed from in vivo measurements made with point sources demonstrate the feasibility of this method.
A singlet oxygen monitor as an in vivo photodynamic therapy dosimeter
Author(s):
S. Lee;
K. Galbally-Kinney;
M. F. Hinds;
J. A. O'Hara;
B. W. Pogue;
A. Liang;
T. Hasan;
S. J. Davis
Show Abstract
In this paper we describe the development and testing of instruments to measure singlet molecular oxygen produced by
the photodynamic process. Singlet oxygen is an active species in photodynamic therapy, and we are developing two
instruments for PDT researchers with the goal of a real-time dosimeter for singlet oxygen. We discuss both an ultrasensitive
point sensor, and an imaging system that provides simultaneous 2D maps of the photosensitizer fluorescence
and the singlet oxygen emission. Results of in vitro tests to characterize the sensors and preliminary in vivo results are
presented.
Bladder cancer detection by fluorescence imaging with Hexvix: analysis and processing of images obtained during high magnification cystoscopy
Author(s):
Blaise Lovisa;
Patrice Jichlinski;
Daniela Aymon;
Bernd-Claus Weber;
Hubert van den Bergh;
Georges Wagnières
Show Abstract
Fluorescence cystoscopy has been recently acknowledged as a useful method to detect early superficial bladder cancer,
even flat lesions. After the instillation of hexaminolevulinic acid (Hexvix) in the bladder for about an hour,
photoactivable porphyrins (PaP), mainly protoporphyrin IX (PpIX) accumulate in the cancerous cells. Although we
observe a selective production of PpIX and an outstanding sensitivity of this method, false positive (FP) lesions
negatively impact its specificity. Carcinogenesis often combines with angiogenesis, and thus changes in vascular
architecture. Therefore, the visualization of the vascular modifications on the fluorescence positive sites is likely to
differentiate false and true positive (TP). New methods including high magnification (HM) cystoscopy are being
investigated by our group, and will yield a reduced number of biopsies and a better characterization of the fluorescence
positive sites. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. Each
observed site is biopsied and described by histopathological analysis. The vascular organization (tortuosity, vascular
loops, vascular area and diameter) of the fluorescence positive sites was characterized in parallel with an in situ visual
grading and a dedicated software procedure. We describe here a simple image processing prototype that classifies the
HM images into two classes, according to their pixel distributions. For that purpose, we developed an algorithm in the
image spatial and frequency domain, so that the vascular architecture could be described objectively and quantitatively.
Hyperspectral two-photon near-infrared cancer imaging at depth
Author(s):
Nikolay S. Makarov;
Jean Starkey;
Mikhail Drobizhev;
Aleksander Rebane
Show Abstract
We present a novel way of optical detection of malignant cancer cell colonies by using multi-wavelength two-photon
excited fluorescence from an environmentally sensitive Styryl-9M dye. We show that the two-photon excited
fluorescence from colonies embedded in a tissue phantom depends on the type of cells as well as on the composition of
the phantoms. We use the ratio between the fluorescence intensities excited at 1100 and 1200 nm to distinguish between
samples containing no cell colonies, samples with colonies of normal cells and samples with cancer cells. The proposed
method is a promising tool for non-invasive deep tissue photodetection diagnostics and for precise localization of
malignant cells.
The design of a robotic multichannel platform for photodynamic therapy
Author(s):
Yida Hu;
Jarod C. Finlay;
Timothy C. Zhu
Show Abstract
A compact robotic platform is designed for simultaneous multichannel motion control for light delivery and dosimetry
during interstitial photodynamic therapy (PDT). Movements of light sources and isotropic detectors are controlled by
individual motors along different catheters for interstitial PDT. The robotic multichannel platform adds feedback
control of positioning for up to 16 channels compared to the existing dual-motor system, which did not have
positioning encoders. A 16-channel servo motion controller and micro DC motors, each with high resolution optical
encoder, are adopted to control the motions of up to 16 channels independently. Each channel has a resolution of
0.1mm and a speed of 5cm/s. The robotic platform can perform light delivery and dosimetry independently, allowing
arbitrary positioning of light sources and detectors in each catheter. Up to 16 compact translational channels can be
combined according to different operational scheme with real-time optimal motion planning. The characteristic of high
speed and coordinating motion will make it possible to use short linear sources (e.g., 1- cm) to deliver uniform PDT
treatment to a bulk tumor within reasonable time by source stepping optimization of multiple sources simultaneously.
Advanced robotic control algorithm handles the various unexpected circumstance in clinical procedure, e.g., positiontorque/
current control will be applied to prevent excessive force in the case of resistance in the fiber or motorized
mechanism. The robotic platform is fully compatible with operation room (OR) environment and improves the light
delivery and dosimetry in PDT. It can be adopted for diffusing optical tomography (DOT), spectroscopic DOT and
fluorescent spectroscopy.
Enhancement of tumor responsiveness to aminolevulinate-photodynamic therapy (ALA-PDT) using differentiation-promoting agents in mouse models of skin carcinoma
Author(s):
Sanjay Anand;
Golara Honari;
Akshat Paliwal;
Tayyaba Hasan;
Edward V. Maytin
Show Abstract
Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is an emerging treatment for cancers. ALA, given as a
prodrug, selectively accumulates and is metabolized in cancer cells to form protoporphyrin IX (PpIX). Targeted local
irradiation with light induces cell death. Since the efficacy of ALA-PDT for large or deep tumors is currently limited, we
are developing a new approach that combines differentiation-inducing agents with ALA-PDT to improve the clinical
response. Here, we tested this new combination paradigm in the following two models of skin carcinoma in mice: 1)
tumors generated by topical application of chemical carcinogens (DMBA-TPA); 2) human SCC cells (A431) implanted
subcutaneously. To achieve a differentiated state of the tumors, pretreatment with a low concentration of methotrexate
(MTX) or Vitamin D (Vit D) was administered for 72 h prior to exposure to ALA. Confocal images of histological
sections were captured and digitally analyzed to determine relative PpIX levels. PpIX in the tumors was also monitored
by real-time in vivo fluorescence dosimetry. In both models, a significant increase in levels of PpIX was observed
following pretreatment with MTX or Vit D, as compared to no-pretreatment controls. This enhancing effect was
observed at very low, non-cytotoxic concentrations, and was highly specific to cancer cells as compared to normal cells.
These results suggest that use of differentiating agents such as MTX or Vit D, as a short-term combination therapy given
prior to ALA-PDT, can increase the production of PpIX photosensitizer and enhance the therapeutic response of skin
cancers.
Improvement of anti-tumor activity of photodynamic therapy through inhibition of cytoprotective mechanism in tumor cells
Author(s):
Dominika Nowis;
Angelika Szokalska;
Marcin Makowski;
Magdalena Winiarska;
Jakub Golab
Show Abstract
Photodynamic therapy (PDT) leads to oxidative damage of cellular macromolecules, including numerous
proteins that undergo multiple modifications such as fragmentation, cross-linking and carbonylation that result
in protein unfolding and aggregation. Several mechanisms are involved in the protective responses to PDT
that include activation of transcription factors, heat shock proteins, antioxidant enzymes and antiapoptotic
pathways. Identification of these cytoprotective mechanisms might result in the design of more effective
combination strategies to improve the antitumor efficacy of PDT. By using various molecular biology
approaches, including microarray-based technologies we have identified genes that are up-regulated
following PDT. Subsequent experiments revealed that some of these gene products can become targets for
the combined therapeutic regimens encompassing PDT and selective small-molecule inhibitors. These
include superoxide dismutase (SOD-2), cyclooxygenase 2 (COX-2), heme oxygenase 1 (HO-1), and proteins
engaged in signaling endoplasmatic reticulum (ER) stress and unfolded protein response (UPR).
Since a major mechanism for elimination of carbonylated proteins is their degradation by
proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to
accumulation of carbonylated proteins in ER, aggravated ER stress and potentiated cytotoxicity towards
tumor cells. Indeed, we observed that incubation of tumor cells with three different proteasome inhibitors,
including bortezomib, MG132 and PSI gave increased accumulation of carbonylated and ubiquitinated
proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells to PDT-mediated
cytotoxicity and augmented antitumor effects of PDT in vivo.
Comparison of ATP level in U937 cells in vitro using two different photosensitizers in photodynamic therapy
Author(s):
Nobue Nakajima;
Norimichi Kawashima
Show Abstract
The effect of PDT on ATP level in the Leukemic monocyte lymphoma cell line (U937) was studied using two different
photosensitizers, PpIX and Hypericin. U-937 cells were irradiated (with and without ALA) and stored at room
temperature. An LED lamp with a maximum peak of 637nm was used for ALA-PDT as a light source. A Na-Li lamp
with a wavelength region of 560-730nm was used for Hypericin-PDT as a light source. We measured cell viability, the
number of viable cells and ATP luminescence. As a result, it is evident that the irradiation time in the most effective PDT
is equal to the time of the highest ATP level in the cells in ALA-PDT. This study suggests that it is possible to obtain
suitable PDT conditions inducing apoptosis by measuring the ATP level in ALA-PDT. On the other hand, ATP level in
Hypericin-PDT was increased with the decreasing in the cell viability. Hypericin-PDT induced apoptosis just after
irradiation.
Comparison of aminolevulinic acid and hexylester aminolevulinate induced protoporphyrin IX fluorescence for the detection of ovarian carcinoma metastases: an experimental study
Author(s):
Manuel Ascencio;
Claudia Regis;
Serge Mordon;
Pierre Collinet
Show Abstract
The present study aimed at comparing the photo detection of peritoneal micrometastases in an ovarian cancer model
following administration of two precursors of protoporphyrin IX (PpIX): aminolevulinic acid (ALA) and hexylester
aminolevulinate (He-ALA).
ALA or He-ALA (100mg/kg) was injected in the peritoneum cavity of 16 rats with induced peritoneal metastases of
ovarian cancer. Two hours later, the tumours were visualized laparoscopically using both white light for standard
exploration and blue light for fluorescence (D-light, Karl Storz, Tuttlingen, Germany). Peritoneal micrometastases were
counted. The distribution of PpIX through the peritoneum was studied on frozen biopsies using fluorescence microscopy
and correlated with pathological findings.
The number of micrometastases detected by the fluorescence blue mode was significantly higher (p<0.05) than with
standard white light for both ALA (235 versus 198) and He-ALA application (248 versus 199). The mean fluorescence
intensity ratio between tumor and normal surrounding tissue was significantly (p< 0.05) higher for He-ALA (1.6±0.1)
compared to ALA (1.4±0.1). Fluorescence microscopy confirmed that the fluorescence remained limited to cancer cells.
Macroscopically fluorescing nodules were histopathology confirmed as malignant.
In conclusion, He-ALA is an excellent precursor for PpIX synthesis giving the highest PpIX fluorescence contrast
between normal and tumoral peritoneum. Imaging with He-ALA improves the detection of peritoneal metastases
comparing to ALA.
Light fractionation increases the efficacy of ALA-PDT but not of MAL-PDT: What is the role of (vascular) endothelial cells?
Author(s):
H. S. de Bruijn;
H. C. de Vijlder;
E. R. M. de Haas;
A. van der Ploeg-van den Heuvel;
B. Kruijt;
D. Poel-Dirks;
H. J. C. M. Sterenborg;
T. L. M. ten Hagen;
D. J. Robinson
Show Abstract
Photodynamic therapy (PDT) using protoporpyrin IX (PpIX) precursors like 5-aminolevulinic acid (ALA) or
methyl-aminolevulinate (MAL) has shown to be effective in the treatment of various skin diseases. Using ALA
we have shown in numerous studies a significantly improved efficacy by applying light fractionation with a
long dark interval. In contrast, in the hairless mouse model, the PDT efficacy using MAL is unaffected by
adopting this approach. More acute edema is found after ALA-PDT suggesting a difference in response of
endothelial cells to PDT.
To investigate the role of endothelial cells, cryo-sections of hairless mouse skin after 4 hours of topical MAL
or ALA application were stained with a fluorescent endothelial cell marker (CD31). Co-localization of this
marker with the PpIX fluorescence was performed using the spectral imaging function of the confocal
microscope. We have also used intra-vital confocal microscopy to image the PpIX fluorescence distribution in
correlation with the vasculature of live mouse skin.
Our results show PpIX fluorescence at depth in cryo-sections of mouse skin after 4 hours of topical
application. Co-localization has shown to be difficult due to the changes in tissue organization caused by the
staining procedure. As expected we found high PpIX fluorescence levels in the epidermis after both MAL and
ALA application using intra-vital microscopy. After ALA application more PpIX fluorescence was found deep in
the dermal layer of the skin than after MAL. Furthermore we detected localized fluorescence in unidentified
structures that could not be correlated to blood vessels or nerves.
Burn, freeze, or photo-ablate?: comparative symptom profile in Barrett's dysplasia patients undergoing endoscopic ablation
Author(s):
Kanwar Rupinder S. Gill;
Seth A. Gross;
Bruce D. Greenwald;
Lois L. Hemminger;
Herbert C. Wolfsen
Show Abstract
Background: There are few data available comparing endoscopic ablation methods for Barrett's esophagus with high-grade
dysplasia (BE-HGD).
Objective: To determine differences in symptoms and complications associated with endoscopic ablation.
Design: Prospective observational study.
Setting: Two tertiary care centers in USA.
Patients: Consecutive patients with BE-HGD
Interventions: In this pilot study, symptoms profile data were collected for BE-HGD patients among 3 endoscopic
ablation methods: porfimer sodium photodynamic therapy, radiofrequency ablation and low-pressure liquid nitrogen
spray cryotherapy.
Main Outcome Measurements: Symptom profiles and complications from the procedures were assessed 1-8 weeks after
treatment.
Results: Ten BE-HGD patients were treated with each ablation modality (30 patients total; 25 men, median age: 69
years (range 53-81). All procedures were performed in the clinic setting and none required subsequent hospitalization.
The most common symptoms among all therapies were chest pain, dysphagia and odynophagia. More patients (n=8) in
the porfimer sodium photodynamic therapy group reported weight loss compared to radio-frequency ablactation (n=2)
and cryotherapy (n=0). Four patients in the porfimer sodium photodynamic therapy group developed phototoxicity
requiring medical treatment. Strictures, each requiring a single dilation, were found in radiofrequency ablactation (n=1)
and porfimer sodium photodynamic therapy (n=2) patients.
Limitations: Small sample size, non-randomized study.
Conclusions: These three endoscopic therapies are associated with different types and severity of post-ablation
symptoms and complications.
Meta-analysis of five photodisinfection clinical trials for periodontitis
Author(s):
Roger C. Andersen;
Nicolas G. Loebel;
Dane M. Andersen
Show Abstract
Photodynamic therapy(PDT) has been demonstrated to effectively kill human periopathogens in vitro. To evaluate the
efficacy of PDT in vivo a series of clinical trials was carried out in multiple centers and populations. Clinical parameters
including clinical attachment level, pocket probing depth and bleeding on probing were all evaluated. All groups
received the standard of care, scaling and root planing, and the treatment group additionally received a single treatment
of PDT. Of the total 309 patients and over 40,000 pockets treated in these 5 trials it was determined that photodynamic
therapy provided a statistically significant improvement in clinical parameters over scaling and root planing alone.
Short and long term stability of the Diomed 630PDT laser evaluated with integrating sphere, power meter, and calorimeter
Author(s):
C. Austerlitz;
D. Campos;
R. Allison;
C. Sheng;
C. Bonnerup;
C. Sibata
Show Abstract
The short and long term stability of the Diomed 630 PDT laser with attached fiberoptic microlens was evaluated by
means of integrating sphere, power meter and a calorimetric system. The calorimeter system was designed as a thermal
mug with absorbing media (dye and water). Both the tip of the irradiation fiber and the detection probe of a
thermocouple thermometer were positioned inside the dye solution and stirred during the measurements. The
calorimetric system yielded measurement results consistent with the other two methods, and similar long term variations
were observed by all methods. With an indicated laser power of 1 W, the detectors' readings ranged from 0.66 to 1.29
W. For short term stability study, the deviation of laser output assessed by integrating sphere, power meter and
calorimetric system were 0.3%, 0.1% and 2.8% with long term deviations of 13%, 7% and 9% respectively. This wide
variation in the laser output implies the needs to establish quality control procedures involving measurements pre and
post PDT procedures. The calorimetric system has been demonstrated to be a powerful tool for clinical laser QA and
maintenance of the calibration factor of the detectors used in this work.
Investigation into the potential of sub-lethal photodynamic antimicrobial chemotherapy (PACT) to reduce susceptibility of meticillin-resistant Staphylococcus aureus (MRSA) to antibiotics
Author(s):
C. M. Cassidy;
R. F. Donnelly;
M. M. Tunney
Show Abstract
In PACT, a combination of a sensitising drug and visible light cause the selective destruction of microbial cells via
singlet oxygen production. As singlet oxygen is a non-specific oxidizing agent and is only present during illumination,
development of resistance to this treatment is thought to be unlikely. However, in response to oxidative stress, bacteria
can up-regulate oxidative stress genes and associated antibiotic resistance genes. The up-regulation of these genes and
potential transfer of genetic material may result in a resistant bacterial population. This study determined whether
treatment of clinically isolated meticillin resistant Staphylococcus aureus (MRSA) strains with sub-lethal doses of
methylene blue (MB) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP)-PACT resulted in reduced
susceptibility to antibiotics and previously lethal PACT. Exposure of strains to sub-lethal doses of photosensitizer in
combination with light had no effect on susceptibility to previously lethal photosensitization. Furthermore, exposure to
sub-lethal concentrations of both photosensitizers caused no significant changes in the minimum inhibitory concentration
(MIC) for each strain tested. Any differences in susceptibility were not significant as they did not cross breakpoints
between resistant and susceptible for any organism or antibiotic tested. Therefore, PACT remains an attractive
alternative option for treatment of MRSA infections.
Synthesis and cellular localization of porphyrinic pigments
Author(s):
Sarah Sareh;
Sarah Kong;
Lenin Parrales;
Anna Jung;
Kara Cross;
Beate Röder;
Meden Isaac;
Ursula Simonis
Show Abstract
To determine factors that govern the uptake preference of photosensitizers in cellular organelles of human
adenocarcinoma cells, diarginyl-dialkoxy- and diarginyl-dimethoxyphenylporphyrins (TPPs) and two of their
corresponding indium(III) complexes were synthesized, characterized and incubated in androgen-sensitive human
prostate adenocarcinoma cells LNCaP. The porphyrins revealed properties that are of importance for phototherapy.
They are water-soluble, have their fourth Q-band absorbing at ≈ 650 nm, are taken up in relatively high concentrations in
LNCaP cells, and are phototoxic. Colocalization and phototoxicity studies revealed that all porphyrins localized
preferentially to the lysosomes and invoked cell death when excited with 650 nm light. Compared to the corresponding
methoxy-substituted TPPs, the diargininyl-dialkoxy-substituted porphyrins localized to a small extent in the
mitochondria. The corresponding In(III) chloride complexes that are slightly less water-soluble were also taken up in the
lysosomes of LnCaP cells. When the TPPs were compared to a pheophorbide derivative recently synthesized in our
laboratory, it was determined that the TPPs have a preference for lysosomal localization, whereas the pheophorbide
derivative co-localized to the mitochondria. Phototoxicity studies revealed that the longer chain dialkoxyTPPs were
more effective in cell killing and induced greater morphological changes typical of apoptotic cell death than the shorter
chain methoxy substituted porphyrins. The In(III) complexes seemed to be the most phototoxic. These results highlight
that the type, nature, and substitution pattern of the chromophore modulate the extent of apoptotic cell death and
influence cellular targeting.
Effects of vascular targeting photodynamic therapy on lymphatic tumor metastasis
Author(s):
B. Fateye;
C. He;
B. Chen
Show Abstract
Vascular targeting photodynamic therapy (vPDT) is currently in clinical trial for prostate cancer (PCa)
treatment. In order to study the effect of vPDT on tumor metastasis, GFP-PC3 or PC-3 xenografts were
treated with verteporfin (BPD) PDT. Vascular function was assessed by ultrasound imaging; lymph node and
lung metastasis were assessed by fluorescence imaging. vPDT significantly reduced tumor blood flow within
30minutes to 2 hours of treatment. Sub-curative treatment resulted in re-perfusion within 2 weeks of treatment
and increased lymph node metastasis. With curative doses, no metastasis was observed.
In order to identify cellular or matrix factors and cytokines implicated, conditioned medium from BPD PDTtreated
endothelial cells was incubated with PC3 cells in vitro. Tumor cell proliferation and migration was
assessed. By immunoblotting, we evaluated the change in mediators of intracellular signaling or that may
determine changes in tumor phenotype. Low sub-curative dose (200ng/ml BPD) of endothelial cells was
associated with ~15% greater migration in PC3 cells when compared with control. This dose was also
associated with sustained activation of Akt at Ser 473, an upstream effector in the Akt/ mTOR pathway that
has been correlated with Gleason scores in PCa and with survival and metastasis in vitro and in vivo. In
conclusion, the study implicates efficacy of PDT of endothelial cells as an important determinant of its
consequences on adjacent tumor proliferation and metastasis.
In vitro and in vivo evaluation of the induced PDT response using a femtosecond laser
Author(s):
C. Grecco;
C. Kurachi;
V. S. Bagnato
Show Abstract
One of the limitations of PDT in the treatment of bulk tumors is the light penetration in biological tissues at the red
spectrum. PDT illumination at short pulsed regime may present a higher light penetration compared to the CW regime,
and potentially a higher volume of induced necrosis. The major goal of this study was an in vitro and in vivo evaluation
of PDT response after illumination using a 630 nm femtosecond laser. Photogem in distilled water solution was
illuminated either with CW or femtosecond laser under the same fluence and fluence rate parameters. Wistar rats
weighting between 280 and 300 g were intravenously photosensitized with Photogem. Thirty minutes after drug
injection, the normal liver was irradiated either with the CW or the femtosecond laser (fluence: 150 J/cm2). The in vitro
results showed that, under the same conditions, the degradation rate evaluated via fluorescence spectroscopy was higher
under femtosecond laser irradiation. Histological analysis of the induced necrosis showed that there was a significant
higher depth of necrosis when the femtosecond laser was used. Based on these results, femtosecond lasers seem to be an
alternative in PDT applications, improving results for the treatment of lesions for which a larger light penetration is
required.
The new application of photosensitization reaction to atrial fibrillation treatment: mechanism and demonstration of non-thermal electrical conduction block with porcine heart
Author(s):
Arisa Ito;
Hiroki Matsuo;
Tsukasa Suenari;
Takuro Kajihara;
Takehiro Kimura;
Shunichiro Miyoshi;
Satoshi Ogawa;
Tsunenori Arai
Show Abstract
We have proposed non-thermal electrical conduction block for atrial fibrillation treatment by the photosensitization
reaction, in which the interval time between the photosensitizer injection and irradiation is less than tenth of that in
conventional way. To study the mechanism of photosensitization reaction-induced electrical conduction block,
intracellular Ca2+ concentration change in rat myocardial cells was measured by fluorescent Ca2+ indicator Fluo-4 AM
with confocal laser microscopy. Measured rapid increase in the fluorescence intensity and a change in cell morphology
indicated that cell membrane damage; that is Ca2+ influx and eventually cell death caused by the photosensitization
reaction. To demonstrate myocardial electrical conduction block induced by the photosensitization reaction, surgically
exposed porcine heart under deep anesthesia was used. The myocardial tissue was paced with a stimulation electrode.
The propagated electrical signals were measured by bipolar electrodes at two different positions. Thirty minutes after
the injection of 5-10 mg/kg Porfimer sodium or Talaporfin sodium, the red laser light was irradiated to the tissue point
by point crossing the measuring positions by the total energy density of less than 200 J/cm2. The electrical signal
conduction between the measuring electrodes in the myocardial tissue was delayed by each irradiation procedure. The
electrical conduction delay corresponded to the block line length was obtained. These results demonstrated the
possibility of non-thermal electrical conduction block for atrial fibrillation treatment by the photosensitization reaction.
Treatment of vulvar/vaginal condyloma by HPV: developed instrumentation and clinical report
Author(s):
N. M. Inada;
C. Kurachi;
J. Ferreira;
E. S. Ribeiro;
O. C. C. Guimarães;
S. M. Quintana;
W. Lombardi;
V. S. Bagnato
Show Abstract
Human papillomavirus (HPVs) are a family of sexually viruses with over 100 different genotypes identified till date.
They are associated in 99% of cervical cancers, with HPV16 found in about 50% of cases. They are a cause of the
second most common female cancer worldwide. PDT may constitute an alternative treatment for condyloma by HPV. In
this work we present the development of a PDT device specifically designed for the treatment of vulvar and vaginal
lesions induced by HPV. This equipment has been used in a clinical protocol and it is optically based on 640 nm LED
(light emitting diodes) arrays. There are three illumination probes available that were anatomically designed for specific
site applications: a 30 mm x 115 mm diffuser cylinder for intravaginal illumination and uniform irradiance of 42
mW/cm2; a 36 mm circular probe with 118 mW/cm2 and a 74 mm circular probe with 57 mW/cm2, both for external
illumination. The 10% aminolevulinic acid cream is topically placed over the lesions and 4-6 hours after the application
the illumination is performed. The illumination time is set depending on the chosen probe and treatment area to achieve a
fluence of 200 J/cm2. In this presentation, the preliminary results of this clinical trial will be presented.
Design and evaluation of excitation light source device for fluorescence endoscope
Author(s):
Hyun Soo Lim
Show Abstract
This study aims at designing and evaluating light source devices that can stably generate light
with various wavelengths in order to make possible PDD using a photosensitizer and diagnosis
using auto-fluorescence. The light source was a Xenon lamp and filter wheel, composed of an
optical output control through Iris and filters with several wavelength bands. It also makes the
inducement of auto-fluorescence possible because it is designed to generate a wavelength band of
380-420nm, 430-480nm, and 480-560nm. The transmission part of the light source was
developed to enhance the efficiency of light transmission. To evaluate this light source, the
characteristics of light output and wavelength band were verified. To validate the capability of this
device as PDD, the detection of auto-fluorescence using mouse models was performed.
Fluorescence diagnosis in tissue injury
Author(s):
Vitória Helena Maciel;
Juliana Ferreira;
Vanderlei S. Bagnato
Show Abstract
Background and Objectives: The paper aim was to evaluate the efficacy of the fluorescence spectroscopy in
the detection of UV-induced skin change of Wistar rats.
Study Design/ Materials and Methods: In a group male Wistar rats, the skin damage was produced by an
UV-C lamp, periodically monitored using the laser-induced fluorescence, until complete healing process.
After determining a characteristic emission band present in the fluorescence spectra of the induced injuries,
the amplitude band monitoring allowed the follow up on the injury and the recovery.
Results: We observed the appearance of two new emission bands more evident at the injury spectra when
compared to the spectrums from normal non-exposed tissue. Following such spectral bands was possible to
observe the establishment and recovery.
Conclusions: The fluorescence spectroscopy is a promising technique in distinguishing between normal and
UV induced skin change helping the evaluation of changes which are irreversible cancer tissue characteristics.
Novel non-thermal atrial fibrillation treatment with photosensitization reaction: possibility of permanent electrical blockade in rat chronic model
Author(s):
Hiroki Matsuo;
Arisa Ito;
Shunichiro Miyoshi;
Kyoko Soejima;
Satoshi Ogawa;
Tsunenori Arai
Show Abstract
We demonstrated a possibility of electrical conduction block by ex vivo and in vivo experiments using rat models to
establish a non-thermal treatment for atrial fibrillation by photosensitization reaction (PR). One hour after the
injection of 2 mg/kg Talaporfin sodium to Wistar rat, the right ventricle (1.4 mmT) was extracted. Paced with a
stimulation electrode, this tissue was placed in a tissue bath and immersed in irrigated Tyrode's solution of 37°C
with 8 μg/ml Talaporfin sodium and the gas mixture bubbling of 95% CO2 and 5% O2. The propagated electrical
signal was measured by two bipolar electrodes. Exciting light of 670 nm in wavelength was irradiated to the tissue
between the bipolar electrodes by the power density of 1 W/cm2. After this irradiation, propagation signal blockade
was obtained and continued up to three hours. Rat atrioventricular (AV) node was employed as a target region for
chronic model. The heart of Wistar rat was surgically exposed. External four-lead electrocardiogram of this rat was
measured. Thirty minutes after the injection of 10 mg/kg Talaporfin sodium to the rat, exciting light of 663 nm in
wavelength was irradiated to the AV node by the power density of 500 mW/cm2 for ten minutes. Acute AV block
was obtained during the irradiation. Two weeks after this procedure, complete AV block was confirmed. The rat
was sacrificed to obtain the tissue specimen. We found that the AV node was replaced by scarring tissue under the
microscopic observation of the specimen. We verified possibility of permanent electrical conduction block using PR.
Uptake of verteporfin by orthotopic xenograft pancreas models with different levels of aggression
Author(s):
Julia O'Hara;
Kimberley S. Samkoe;
Alina Chen;
P. Jack Hoopes;
Imran Rizvi;
Tayyaba Hasan;
Brian W. Pogue
Show Abstract
Pancreatic cancer is an aggressive disease with a poor prognosis, usually treated with chemoradiation therapy.
Interstitial photodynamic therapy is a potentially effective adjuvant treatment that is under development. In the current
study, two orthotopic pancreatic cancer models (AsPC-1 and Panc-1), have been characterized with respect to growth
rates, morphology and liposomal drug (Verteporfin) uptake and distribution in SCID mice. Fluorescence of Verteporfin
was measured in liver and tumor in vivo using a PDT fluorescence dosimeter with measurements taken before and up to
one hour after tail vein injection. Fluorescence reached a plateau by about 15 minutes and did not decrease over the first
hour. At time points from 15 minutes to 24 hrs, the internal organs (kidney, spleen, pancreas, tumor, muscle, lung, liver,
and skin were excised and scanned on a Typhoon imager. The ratio of fluorescence in tumor versus normal tissues was
analyzed with image processing, calculated at each time point and compared to in vivo results. Tissue distribution of
Verteporfin in relation to functional vasculature marked by DiOc7 was carried out on frozen sections. Final analysis will
result in determination of the ideal time point to administer light to achieve maximum tumor destruction while
preserving normal tissue.
Development of a widefield reflectance and fluorescence imaging device for the detection of skin and oral cancer
Author(s):
S. Pratavieira;
P. L. A. Santos;
V. S. Bagnato;
C. Kurachi
Show Abstract
Oral and skin cancers constitute a major global health problem that cause great impact in patients. The most common
screening method for oral cancer is visual inspection and palpation of the mouth. Visual examination relies heavily on
the experience and skills of the physician to identify and delineate early premalignant and cancer changes, which is not
simple due to the similar characteristics of early stage cancers and benign lesions. Optical imaging has the potential to
address these clinical challenges. Contrast between normal and neoplastic areas may be increased, distinct to the conventional
white light, when using illumination and detection conditions. Reflectance imaging can detect local changes in
tissue scattering and absorption and fluorescence imaging can probe changes in the biochemical composition. These
changes have shown to be indicatives of malignant progression. Widefield optical imaging systems are interesting because
they may enhance the screening ability in large regions allowing the discrimination and the delineation of neoplastic
and potentially of occult lesions. Digital image processing allows the combination of autofluorescence and reflectance
images in order to objectively identify and delineate the peripheral extent of neoplastic lesions in the skin and oral cavity.
Combining information from different imaging modalities has the potential of increasing diagnostic performance, due to
distinct provided information. A simple widefiled imaging device based on fluorescence and reflectance modes together
with a digital image processing was assembled and its performance tested in an animal study.
Multifunctional ORMOSIL and PAA nanoparticles
Author(s):
Anurag Gupta;
K. V. R. Rao;
Paula Pera;
Shouyan Jason Wang;
Joseph R. Missert;
Tymish Ohulchanskyy;
Indrajit Roy;
Janet Morgan;
Paras N. Prasad;
Raoul Kopelman;
Ravindra K. Pandey
Show Abstract
Various problems arising during molecular imaging of different fluoroprobes and metabolites used in PDT can be
circumvented by focusing on multifunctional therapy agents. Thus an effective photo sensitizer coupled with other
useful roles to play in PDT treatment make nanoparticles as a good vehicle for different delivery assuming
multifunctional roles not only in PDT but also as therapeutic agents for targeted delivery. A new approach is the
involving use of 100 nm NPs as photo sensitizers and/or imaging agents. In our Lab., we employ two such NPs and are
ORMOSIL (organically Modified Silica) and PAA (Polyacrylamide) which are found to be biologically very safe
without disturbing the therapeutic value. The size of the nanoparticles determined by TEM and Dynamic Light
Scattering are ~30 nm. These NPs are taken up in conjunction with cyanine dye at near infra red as it has been reported
in literature that encapsulated NPs shows very low singlet oxygen production compared with the post-loaded NPs though
the reasons are not yet clear. Therefore, we investigated the idea of post-loading or adsorbing vis-a-vis encapsulation.
Comparative in vivo study of precursors of PpIX (ALA and MAL) used topically in photodynamic therapy
Author(s):
Raquel Ferreira Rego;
Natalia Mayumi Inada;
Juliana Ferreira;
Fernando Manuel Araújo-Moreira;
Vanderlei Salvador Bagnato
Show Abstract
The efficacy of Photodynamic Therapy (PDT) combined with aminolevulinic acid (ALA) or methyl
aminolevulinate (MAL) in treatment of cancer has been studied for over ten years. However, there is no
established dose for the topical use of these drugs in PDT. The purpose of this study was the comparison
of induced PDT response of ALAsense (5-aminolevulinic acid - ALA) and Metvix (methyl
aminolevulinate - MAL). Depth of necrosis induced by PDT was analyzed in normal liver of male Wistar
rats, using different light doses and topical application of both PpIX precursors - ALA and MAL. PDT
was performed with a diode laser at 630 nm with different doses of light (20, 50, 100 and 200 J/cm2), and
intensity of 250 mW/cm2. Depth of necrosis analysis was used to calculate the threshold dose for each
drug. The results showed that MAL-PDT presented a better response than ALA-PDT, mainly due to
formulation differences. Moreover, the ability of the ALA PpIX production was more efficient.
Photodynamic therapy for difficult-to-treat basal cell carcinomas: Do poorly responding BCCs lack accumulation of protoporphyrin IX after ALA/MAL application?
Author(s):
Carin Sandberg;
John Paoli;
Christina B. Halldin;
Martin Gillstedt;
Olle Larkö;
Ann-Marie Wennberg;
Marica B. Ericson
Show Abstract
Photodynamic therapy (PDT) using topical application of aminolevulinic acid (ALA) and methylaminolevulinate (MAL)
has become a popular therapeutic method for the treatment of non-melanoma skin cancers such as basal cell carcinomas
(BCCs); however, the treatment response varies. An important question is if BCCs which respond poorly to PDT lack
accumulation of protoporhyrin IX (PpIX) after ALA/MAL application. In connection to PDT, fluorescence diagnostics
(FD) can be performed to detect PpIX within human skin. We investigated fluorescence images from 22 patients with 35
BCCs. They were evaluated with respect to the fluorescence contrast based on image analysis, which was considered to
be a tool to non-invasively measure the PpIX-concentration. As expected the fluorescence contrast between tumor and
normal skin was elevated after MAL-application; although no correlation between low fluorescence contrast and lack of
treatment response could be observed. In a former study, we have also investigated the transdermal penetration of ALA
and MAL in 27 BCCs in vivo using a microdialysis technique. In 15 of 16 BCCs in which the microdialysis catheter was
located superficially (i.e. at a depth of less than 1 mm), therapeutic drug concentrations were detected;.however, in the
11 lesions with a deeper catheter location (below 1 mm) drug concentrations above the detection limit of the system were
only obtained in 6 lesions (p=0.026). No difference between the transdermal penetration of MAL and ALA could be
seen. Conclusions: Lack of PpIX fluorescence cannot entirely explain why some BCCs don't respond to PDT, but
inadecuate concentrations within the full thickness of the tumor may play a role as microdialysis has shown.
Downregulation of the autophagy protein ATG-7 correlates with distinct sphingolipid profile in MCF-7 cells sensitized to photodamage
Author(s):
Duska Separovic;
Ameeta Kelekar;
Adi L. Tarca;
Jacek Bielawski;
David Kessel
Show Abstract
The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell
death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and
their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the
autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in
ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h)
and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced,
whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown
cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.
Synthesis, characterization, and subcellular localization studies of amino acid-substituted porphyrinic pigments
Author(s):
Lisa van Diggelen;
Hnin Khin;
Kip Conner;
Jenny Shao;
Margaretta Sweezy;
Anna Hyewon Jung;
Meden Isaac;
Ursula Simonis
Show Abstract
Stopping cancer in its path occurs when photosensitizers (PSs) induce apoptotic cell death after their exposure to light
and the subsequent formation of reactive oxygen species. In pursuit of our hypothesis that mitochondrial localizing PSs
will enhance the efficacy of the photosensitizing process in photodynamic therapy, since they provoke cell death by
inducing apoptosis, we synthesized and characterized tetraphenylporphyrins (TPPs) that are substituted at the paraphenyl
positions by two amino acids and two fluoro or hydroxyl groups, respectively. They were prepared according to
the Lindsey-modified Adler-Longo methodology using trifluoromethanesulfonylchloride (CF3SO2Cl) as a catalyst
instead of trifluoroacetic acid. The use of CF3SO2Cl yielded cleaner products in significantly higher yields. During the
synthesis, not only the yields and work-up procedure of the TPPs were improved by using CF3SO2Cl as a catalyst, but
also a better means of synthesizing the precursor dipyrromethanes was tested by using indium(III) chloride. Column
chromatography, HPLC, and NMR spectroscopy were used to separate and characterize the di-amino acid-dihydroxy, or
difluoro-substituted porphyrins and to ascertain their purity before subcellular localization studies were carried out.
Studies using androgen-sensitive human prostate adenocarcinoma cells LNCaP revealed that certain amino acid
substituted porphyrins that are positively charged in the slightly acidic medium of cancer cells are very useful in
shedding light on the targets of TPPs in subcellular organelles of cancer cells. Although some of these compounds have
properties of promising photosensitizers by revealing increased water solubility, acidic properties, and innate ability to
provoke cell death by apoptosis, the cell killing efficacy of these TPPs is low. This correlates with their subcellular
localization. The di-amino acid, di-hydroxy substituted TPPs localize mainly to the lysosomes, whereas the di-fluoro-substituted
TPPs are trapped in the plasma membrane. Only a pheophorbide derivative recently synthesized in our
laboratory localized to the mitochondria of LNCaP cells, which are at the center of cell death as is reflected in their key
role during apoptosis, thus reassuring our attempts toward rational drug design.
Effect of hyperthermia on PDT and imaging
Author(s):
Avinash Srivatsan;
K. V. R. Rao;
Yihui Chen;
Yanfang Wang;
Carrie Batt;
Janet Morgan;
Arindam Sen;
Elizabeth Repasky;
Ravindra K. Pandey
Show Abstract
Photodynamic Therapy (PDT) is emerging as a successful tool to treat both malignant and benign tumors. It involves the
interaction of a photosensitizer which upon activation by the appropriate light dose, leads to a cytotoxic and vasculotoxic
photodynamic reaction. Improvements in PDT in areas such as the delivery and selectivity of photosensitizers,
light-delivery and overall efficacy have helped to increase its attractiveness as an option for therapy. For optimizing the
PDT treatment by a "see and treat approach," we have developed a number of tumor avid photosensitizers (PS) namely
HPPH-Cyanine dye conjugates or other compounds (Iodinated photosensitizers) which have the ability for Optical and/or
PET imaging as well as being effective photosensitizers for treatment. Hyperthermia refers to various techniques of heat
application which may be delivered as a single modality or as part of an adjunct treatment option to the existing cancer
therapies. Depending upon the temperature range used, hyperthermia might either directly induce cell kill or enhance the
efficacy of other treatment modalities. Hyperthermia increases blood flow within the body, which may allow for higher
dose delivery of photosensitizers with subsequent increased therapeutic efficacy of PDT. Hyperthermia could also
increase the sensitivity of molecular imaging. The use of multifunctional photosensitizers for imaging and PDT is an
emerging area and we have developed a few such agents in our lab. We wish to explore the use of hyperthermia to
improve the use of such multifunctional photosensitizers from the point of view of imaging and/or therapy.
Hyperthermia can be performed either as a whole-body mode or as localized mode. Our goal is to see which of the two
heating modalities offers us better outcome.
Photodynamic effects of Photodithazine on cervical cancer model
Author(s):
Sei Jun Han;
Lan Ying Wen;
Su Mi Bae;
Young-Seon Hong;
Hong-Seok Jang;
Jeong Whan Lee;
Woong Shick Ahn
Show Abstract
Photodynamic therapy (PDT) has been reported to be effective for treating various tumors and induce apoptosis in many
tumor cells. In this study, we examined a biological significance of PDT with a new photosensitizer, Photodithazine, in a
cervical cancer model using TC-1 cells. Also, to see the accumulation level of Photodithazine in tumours, we measured
the concentration of Photodithazine at indicated time points in tumours of the mice injected with Photodithazine. When
the tumor bearing mice were exposed to varied doses of Photodithazine with laser irradiation (300 J/cm2), retarded
tumor growth was significant in mice treated with PDT, as compared to the control group. On the other hand, in the
uptake of Photodithazine experiments showed that the highest accumulation of Photodithazine in tumors was shown at
0·5 hr after injection and its concentration was decreased. Photodithazine keeps high concentration values during the first
day, Photodithazine showed a rapid clearance from sera as it is relatively long kept by tumor tissue. Taken together, we
propose that PDT after the application of Photodithazine may be effective in the mice system.
Effectiveness in total reduction of Candida albicans promoted by PDT with hypocrellin B:lanthanum
Author(s):
Daniel J. Tofolli;
Renato A. Prates;
Martha S. Ribeiro;
Nilson D. Vieira Jr.;
M. C. E. Hashimoto;
Lilia C. Courrol
Show Abstract
In this work we described the potentiality of the Hypocrellin B (HB) modified with the presence of lanthanum (La3+)
ions, in eliminate Candida albicans in suspension. The results showed that the presence of lanthanum ions promotes a
red shift of the HB absorption band and an enhancement in singlet oxygen quantum yield in 32%. Also in this work we
obtained that the best molar ration between HB and La concentrations was 1:2. No photobleaching was observed in our
experimental conditions. Antimicrobial activity was studied exciting C. albicans suspension with a 460 nm LED and a
660 nm laser both with 330 mW/cm2 irradiance. Best irradiation time, PS concentration and ROS production profile
were determined showing that using 460 nm LED with 10 μM of PS, only 30 s of irradiation time was sufficient to
reduce 100 % C. albicans colonies.
Conjugate systems using delocalized cationic dyes as a carrier of photosensitizers to mitochondria
Author(s):
Youngjae You;
Ethel J. Ngen;
Pallavi Rajaputra
Show Abstract
Following Photofrin, the first generation photosensitizer, several second generation photosensitizers have been
developed with improved characteristics. More recently, third generation photosensitizers are proposed to achieve higher
selectivity toward cancer cells/ tumor tissue. Elevated mitochondrial membrane potential of malignant cells has tested as
a tool for preferential uptake of certain photosensitizers to cancer cells. In the same line, we designed new conjugate
systems where delocalized cationic moiety delivers a photosensitizer to mitochondria. To prove our concept, two
prototype conjugates (TPP-Rh and TPP-AO) were prepared using two cationic dyes (Rhodamine and Acridine Orange)
and a photosensitizer (tetraphenylporphyrin, TPP). The two conjugates generated singlet oxygen quite well.
Interestingly, the two conjugates showed higher cellular uptake by mover than 8 times than TPP-OH as well as higher
phototoxicity. In particular, TPP-Rh showed closer localization pattern to mitochondria than TPP-OH.
Modulation of COX2 and hTERT expression by photodynamic therapy in human colon cancer cells
Author(s):
Christine Li Miu Ngan Yow;
Ellie Shihng Meir Chu
Show Abstract
Photodynamic therapy (PDT) was employed as a cancer therapy with photosensitizer (PS)-loaded cancer cells,
eradicated by the reactive oxygen species after light activation. Cyclo-oxygenase 2 (COX2) is an enzyme expressed in
80% of colon adenocarcinoma and is one of the targets for effective cancer treatment. There is also uprising evidence
that the human telomerase reverse transcriptase (hTERT), a catalytic component of telomerase, is reported as a promising
indicator for monitoring cancer treatment.
In this study, NPe6 mediated PDT on COX2 induced apoptosis in HT-29 was investigated. The cell cycle changes was
analysed by flow cytometry and the hTERT expression at pre and post PDT was evaluated at transcription level by
Taqman real time PCR.
NPe6-PDT in HT-29 cells demonstrated anti-proliferating effect in a drug and light dose dependent manner. LD50 was
achieved at 16μg/mL and 2J/cm2 at 4 hour-post treatment with a significant down-regulation of COX2 expression at
LD30 and LD50 by immunohistochemical staining (IHC) (p<0.05, One-Way ANOVA). Membrane blebbing was detected in over 60% of cells. 35.2% of treated cells arrested in S-phase at LD50 after 24 hours by flow cytometry. A 0.25- and
0.6-fold down-regulation of hTERT mRNA expression was achieved at LD30 and LD50 respectively by TaqMan real-time
PCR.
To summarize, NPe6 mediated PDT down-regulated COX2 expression and triggered cell apoptosis. The hTERT can
serve as an indicative marker for monitoring NPe6-PDT cancer treatment efficacy.
Fractionated PDT with 5-aminolevulinic acid: effective, cost effective, and patient friendly
Author(s):
Hannah C de Vijlder;
Tom A. Middelburg;
Henriette S. de Bruijn;
Dominic J. Robinson;
H. A. Martino Neumann;
Ellen R. M. de Haas
Show Abstract
PDT with ALA and MAL is established as a relatively effective treatment for non-melanoma skin cancer and premalignancies.
PDT is often repeated, because a single treatment gives poor long term results. Preclinical studies showed
that ALA-PDT applying a fractionated illumination scheme with a small first light fraction and a second larger light
fraction separated by a dark interval of two hours resulted in a significant increase in efficacy. Whereas the efficacy was
not enhanced by fractionating MAL-PDT, indicating that ALA-PDT mechanism is not the same as MAL-PDT
mechanism. The increase in efficacy using fractionated PDT was confirmed clinically. A randomized comparative
clinical study comparing fractionated ALA-PDT versus non-fractionated ALA-PDT in the treatment of superficial basal
cell carcinoma showed a significant higher response rate in the lesions treated with fractionated ALA-PDT after a
follow-up of one year ( p<0.002, log-rank test). The five year follow-up is studied at moment. So far the complete
response in the group treated with fractionated ALA-PDT seems to be only a few percentages lower compared to the one
year follow-up. Besides the gain in response rate, fractionated ALA PDT is cost effective. ALA gel is less expensive
than the commercially available MAL (Metvix) and moreover fractionated ALA-PDT takes one treatment day, instead
of two treatment days using the Metvix treatment protocol (two MAL-PDT treatments separated by one week), both
reducing direct and indirect costs and the burden to the patient.
Loss of intercellular adhesion leads to differential accumulation of hypericin in bladder cancer
Author(s):
S. Sasidharan Lucky;
Ramaswamy Bhuvaneswari;
William W. L. Chin;
Weber K. O. Lau;
Malini C. D. Olivo
Show Abstract
Photodynamic diagnosis (PDD) exploits the photoactive nature of certain compounds, namely photosensitizers, in order
to enhance the visual demarcation between normal and neoplastic tissue. Hypericin is one such potent photosensitizer
that preferentially accumulate in neoplastic tissue, and fluoresce in the visible spectrum when illuminated with light of an
appropriate wavelength. In our study, we investigated the role of E-cadherin in the selective permeation of hypericin in
bladder cancer tissues. Clinical studies were done on a series of 43 histologically graded bladder cancer biopsy
specimens, obtained from 28 patients who received intravesical instillations with 8μM hypericin solution for at least 2
hours. Immunohistochemical staining was used to assess the expression of E-cadherin, in the cryosectioned tissues.
Hypericin uptake was examined by fluorescence microscopy. Immunohistochemical staining showed a clear expression
of E-cadherin along the urothelial lining of the normal and pre-malignant tissues. Partial expression of these cell
adhesion molecules were still observed in malignant tissues, however there was a loss of expression to variable extends
along the urothelium. Thus, loss of intercellular adhesion can be associated with enhanced hypericin permeation through
paracellular diffusion.
Combination of angiogenesis inhibitors increases the anti-tumor efficacy of photodynamic therapy in a human bladder tumor xenograft model
Author(s):
Ramaswamy Bhuvaneswari;
Yik Yuen Gan;
Patricia S. P. Thong;
William Wei Lim Chin;
Khee Chee Soo;
Malini Olivo
Show Abstract
Photodynamic therapy (PDT) is a standard treatment for various malignant and non-malignant conditions. Though
therapeutic responses are encouraging, recurrences have been noted, as one of the limitations of PDT is treatment-induced
hypoxia that triggers angiogenesis. The present study evaluates the use of angiogenic inhibitors Avastin, that
targets vascular endothelial growth factor (VEGF) and Erbitux that targets epidermal growth factor receptor (EGFR)
with PDT in an in vivo bladder carcinoma xenograft. Tumor bearing mice were assigned to 6 different categories:
control, PDT only, Avastin + Erbitux, PDT + Avastin, PDT + Erbitux and PDT + Avastin and Erbitux. Treated and
control tumors were monitored for recurrence for up to 90 days. VEGF and EGFR expression was detected in the tumor
tissue. Migratory assay was performed to establish the inhibitory effect of the angiogenesis agents. Using confocal laser
endomicroscopy, the tumor microvasculature was assessed. Tumors treated with the combination therapy of PDT +
inhibitors showed significantly greater response compared to control and PDT only treated group. Combination therapy
treated tumors also showed the most post-treatment damage with reduced tumor vasculature. These results demonstrate
that the combination of PDT with inhibitors that target different angiogenesis pathways can improve tumor control.
Multiplex infectious disease microarrays: STAT serology on a drop of blood
Author(s):
Tom Ewart;
Mark Tarnopolsky;
Steve Baker;
Sandeep Raha;
Yuen-Yee Wong;
Kathy Ciebiera
Show Abstract
New and resurgent viral and antibiotic-resistant bacterial diseases are being encountered worldwide. The US CDC now
ranks hospital acquired infections among the top 10 leading causes of death in the US, costing $20 billion annually. Such
nosocomial infections presently affect 5% - 10% of hospitalized patients leading to 2 million cases and 99,000 deaths
annually. Until now, assays available to mount comprehensive surveillance of infectious disease exposure by biosecurity
agencies and hospital infection control units have been too slow and too costly. In earlier clinical studies we have
reported proteomic microarrays combining 13 autoimmune and 26 viral and bacterial pathogens that revealed
correlations between autoimmune diseases and antecedent infections. In this work we have expanded the array to 40
viruses and bacteria and investigated a suspected role of human endogenous retroviruses in autoimmune neuropathies.
Using scanning laser imaging, and fluorescence color multiplexing, serum IgG and IgM responses are measured
concurrently on the same array, for 14 arrays (patient samples) per microscope slide in 15 minutes. Other advantages
include internal calibration, 10 μL sample size, increased laboratory efficiency, and potential factor of 100 cost
reduction.
A new paradigm for photodynamic therapy: coherent control
Author(s):
Di Yang;
Janne Savolainen;
Aliakbar Jafarpour;
Daan Sprünken;
Jennifer L. Herek
Show Abstract
Photodynamic therapy (PDT) is a treatment based on the interaction of light, photosensitizing agents and tissue oxygen.
The light delivery in PDT is usually optimized by controlling the intensity, the spectrum, and/or the dosage of excitation
light. In this paper, we introduce a novel method that aims to improve the efficiency of PDT by controlling the phase of
the excitation light, an important and so far neglected parameter. This coherent control approach utilizes the coherence
properties of light-matter interaction and aims to manipulate the quantum interferences between various available
reaction pathways. In general, an outcome of a photochemical reaction can be optimized by enhancing the desired
reaction pathways and suppressing other unwanted pathways. Such optimizations can be done by appropriate tailoring of
the electric field profile of a broadband coherent excitation light, i.e. ultrafast laser pulse. Here, we used a femtosecond
laser source with adaptive pulse shaping together with a molecular feedback in a learning loop to search for and
synthesize such 'smart' laser pulses. Our control objective is to enhance the triplet yield of a model photosensitizer zinc
phthalocyanine (ZnPc), which then leads to enhancement of the overall PDT process. We use two coherent control
schemes where we optimize the ratio between the excited singlet state (S) and triplet state (T) ZnPc molecules both ways
(S/T and T/S). We demonstrate a control of 15% over the triplet yield between the found best and the worst pulse shapes.
Our preliminary results show that phase shaping can indeed be used in manipulating photosensitizer photophysics and
correspondingly the yield of singlet oxygen.
Effect of verteporfin-PDT on the Notch signaling pathway in cholangiocarcinoma (CCA) cell lines
Author(s):
Virginie Cerec;
Fausto Andreola;
Stephen P. Pereira
Show Abstract
Accumulating preclinical and clinical evidence supports a pro-oncogenic function for Notch signaling in several solid
tumors. Therefore, Notch inhibitory agents, such as gamma-secretase inhibitors (GSI), are being investigated as cancer
therapeutic agents and a potential adjuvant to conventional chemo/radiotherapy. To date, no in vitro data are available on
the cellular response and effect of either photodynamic therapy (PDT) or GSI on human cholangiocarcinoma (CCA).
Consequently, we aimed to study the: (i) constitutive expression of Notch signaling pathway in CCA cell lines; (ii)
response to Verteporfin-PDT and to GSI, as single agents on CCA cell lines; (iii) effect of Verteporfin-PDT on Notch
signaling pathway expression.
Expression of Notch signaling components was studied in two cholangiocarcinoma cell lines, HuCCT1 and
TFK-1 (intra- and extrahepatic, respectively). No difference in basal expression of Notch1, 2 and Jagged1 was observed
in either cell line. In contrast, Notch3 was found to be weakly and highly expressed in HuCCT1 and TFK-1 cells,
respectively - supporting our recent microarray data which showed Notch3 overexpression in biliary brushings from
patients with extrahepatic CCA. HuCCT1 and TFK-1 differentially responded to Verteporfin-PDT treatment; preliminary
data showed no clear effect of GSI on proliferation/apoptosis in either cell line following short exposure (6 and 24h).
Following Verteporfin-PDT, Notch1, 2 and Jagged-1 expression was down-regulated in both cell lines, while Notch3
expression was unaffected in HuCCT1 cells and down-regulated in TFK-1 cells. The Notch signaling pathway could
represent a potential target for combination therapy in CCA treatment.
Development of novel bifunctional agents with and without tumor imaging ability
Author(s):
Manivannan Ethirajan;
Michael Williams;
Norm Angelino;
Joseph R. Missert;
Ravindra K. Pandey
Show Abstract
Purpurinimides, derived from chlorophyll-a, are tumor avid, stable in vivo and show a strong absorption in the near IR
region (700 nm) with a high singlet oxygen (>50%) producing efficiency. Such a characteristic could be useful in
treating large and deeply seated tumors by photodynamic therapy (PDT). These long wavelength photosensitizers can be
used as vehicles to deliver the imaging agents to tumors. Therefore, by linking suitable tumor imaging moiety with
purpurinimide could produce a novel bifunctional agent, which could possess the desired photo physical properties for
both tumor detection as well as photodynamic therapy. Such image guided therapy would represent an archetype for
cancer treatment.
In vitro and in vivo studies of new photoluminescent oxygen sensors for non-invasive intravascular pO2 measurements
Author(s):
Patrycja Nowak-Sliwinska;
Eddy Forte;
Hubert van den Bergh;
Georges Wagnières
Show Abstract
The concentration of oxygen and its rate of consumption are important factors playing a role in PDT and radiotherapy.
One of the methods for measuring the tissular oxygen partial pressure (pO2) is based on the use of luminophores
presenting an oxygen-dependent quenching of their phosphorescence. The time-resolved luminescence spectroscopy of
palladium (PdTCPP) or ruthenium (RuDPP) porphyrin complexes is used for this purpose. Unfortunately, these
porphyrin derivatives are phototoxic and leak rapidly out of the blood vessels, making them unsuitable for measuring
tissular and or intravascular pO2.
Therefore, this research aimed at developing and testing new biocompatible, non-phototoxic oxygen sensors based on
palladium complexes incorporated into oxygen permeable, polysaccharide-based nanoparticles appropriate for noninvasive
in situ and in vivo measurements of the pO2.
In vitro studies, performed with an optical fiber-based time-resolved spectrophotometer, showed that the incorporation of
such pO2 probes in nanovectors reduces their sensitivity to oxygen as well as their photobleaching by less than one order
of magnitude.
However, in vivo biocompatibility studies performed on the chick's embryo chorioallantoic membrane (CAM) model
demonstrated that the luminescence of those oxygen probes tends to be heterogeneously distributed within the
vasculature. In addition, these probes induce a 'clumping tendency', resulting in a more or less decreased viability of the
embryos.
Effect of verteporfin-PDT on epithelial growth factor receptor (EGFR) signaling pathway in cholangiocarcinoma cell lines
Author(s):
Fausto Andreola;
Virginie Cerec;
Stephen P. Pereira
Show Abstract
EGFR, a member of the ERBB family, plays a pivotal role in carcinogenesis. EGFR overexpression is
implicated in DNA repair and synergistic interactions between EGFR-targeting drugs and conventional
chemo/radiotherapy have been reported in preclinical studies for different cancers but not
cholangiocarcinoma (CCA). To date there are no in vitro data available on the cellular response and
effect of either photodynamic therapy (PDT) or EGFR-targeting drugs on CCA. Therefore, we aimed
to study the: (i) response to Verteporfin PDT and to EGFR-targeting drugs, as single agents; (ii) effect
of PDT on ERBBs expression, phosporylation status and activation of its signaling pathways; (iii)
response to combination of PDT and EGFR-targeting agents.
We showed that two cholangiocarcinoma cell lines (HuCCT1 and TFK1 cells, intra- and extrahepatic,
respectively) differentially respond to verteporfin-PDT treatment and are resistant to EGFR-targeting
agents. A constitutive activation of EGFR in both cell lines was also observed, which could partly
account for the observed resistance to EGFR-targeting drugs. In addition, verteporfin-PDT induced
further phosphorylation of both EGFR and other Receptor Tyrosine Kinases. Mitochondria-independent
apoptosis was induced by PDT in both CCA cell lines; in particular, PDT modulated the
expression of members of the Inhibitor of Apoptosis (IAP) family of proteins. Interestingly, there was a
PDT-induced EGFR nuclear translocation in both cell lines; co-treatment with either an EGFR-inhibitor
(Cetuximab) or a nuclear import blocking agent (Wheat Germ Agglutinin) had an additive
effect on PDT cell killing, thus implying a role of EGFR in repairing the potential PDT-induced DNA
damage.
Catheter-based complex swept source optical coherence tomography for biomedical imaging
Author(s):
Youxin Mao;
Costel Flueraru;
Shoude Chang
Show Abstract
We demonstrate a catheter-based complex swept-source optical coherence tomography (SS-OCT) system using a 3x3
Mach-Zehnder quadrature interferometer and an ultra-small optic probe. Design and fabrication of fiber lens for ultrasmall
optic probes are presented first. We compare in detail measured performance with expected theoretical
performance. Then, we present a 3x3 Mach-Zehnder quadrature interferometer to acquire a complex interferometric
signal for SS-OCT. We introduce an unbalanced differential detection method to improve the overall utilization of
optical power and provide simultaneous access to the complementary phase components of the complex interferometric
signal. No calculations by trigonometric relationships are needed. We compare the performance for our setup to that of a
similar interferometer with a commonly used balanced detection technique. We demonstrate complex conjugate artifact
suppression of 27 dB obtained in our swept-source optical coherence tomography using our unbalanced differential
detection. OCT in vivo and ex vivo images shown in this presentation indicate that our catheter-based complex SS-OCT
system is capable for imaging of biomedical tissues and inside organs for human, small animals and big animals.
Detection and diagnosis of human oral cancer using hypericin fluorescence endoscopic imaging interfaced with embedded computing
Author(s):
Patricia S. P. Thong;
Malini Olivo;
Feng Lin;
Hock-Soon Seah;
Stephanus S. Tandjung;
Kemao Qian;
William W. L. Chin;
Ramaswamy Bhuvaneswari;
Kent Mancer;
Khee-Chee Soo
Show Abstract
Oral cancers are currently diagnosed using white light endoscopy and histopathology. However, oral tumours are mostly
superficial and can be difficult to visualise. Here we present the use of hypericin with fluorescence endoscopy and laser
confocal fluorescence endomicroscopy interfaced with embedded computing for the diagnosis of oral cancers.
Fluorescence imaging of oral lesions was carried out in the clinic using a fluorescence endoscope. The images were
analyzed to extract the red to blue (R/B) ratios to discriminate between tissue types. The results showed that the R/B
ratio is a good image parameter to discriminate between normal, hyperplastic and malignant oral tissue. We are also
developing an embedded, real-time computing system interfaced to a fluorescence endomicroscope for 3D visualization
of tumors, where synchronization of cross-sectional image grabbing and Z-depth scanning is realized through
programming a Field-Programmable Gate Array. In addition to the programming task, a proprietary control circuit has
been developed for the automated 3D reconstruction of fluorescence sections; and preliminary results from fluorescent
samples have demonstrated the potential of this system for real-time in vivo 3D visualization of tumours. This will
ultimately enable same-day clinical diagnosis to be achieved and further enhance the clinical usefulness of fluorescence
diagnostic imaging.
Immediate and long-term efficacy and safety of photodynamic therapy with Photolon (Fotolon): a seven-year clinical experience
Author(s):
Yuri P. Istomin;
Michael A. Kaplan;
Siarhei V. Shliakhtsin;
Tatsiana P Lapzevich;
Dmitriy A. Cerkovsky;
Ludmila N. Marchanka;
Alexander S. Fedulov;
Tatsiana V. Trukhachova
Show Abstract
The purpose of the present study was to summarize data on the long-term efficacy of photodynamic therapy (PDT) with
Photolon in patients with malignant tumors of various types and localizations. The data obtained show that PDT with
Photolon is a highly effective therapeutic modality for the treatment of skin tumors, cervical intraepithelial neoplasias,
lung cancers, disseminated forms of melanoma, primary and metastatic brain tumors, several ophthalmologic diseases.
This paper provides a review of most illustrative studies of the application of PDT with Photolon for the treatment of
different oncological and non-oncological diseases performed in leading clinical centers of the Republic of Belarus and
Russia.
A Monte Carlo model of detected singlet oxygen luminescence and photosensitizer fluorescence during ALA-PDT of skin
Author(s):
Baochang Liu;
Thomas J. Farrell;
Michael S. Patterson;
Mark T. Jarvi;
Brian C. Wilson
Show Abstract
Singlet Oxygen (1O2) Luminescence Dosimetry (SOLD) and fluorescence photobleaching are being investigated
as dosimetric tools for clinical PDT. Both have been applied during superficial ALA-PDT of normal skin and
skin cancers. The interpretation of fluorescence and SOLD data is complicated by the non-uniform distribution
and bleaching of PpIX and the absorption and scattering of light in the skin. The aim of the present work was
to tackle these challenges using Monte Carlo (MC) simulations. Skin was modeled as a three-layer semi-infinite
medium with uniform optical properties in each layer. The initial depth-dependent distribution of PpIX was an
exponential decay and, after the delivery of each treatment fluence increment, standard photochemical reaction
kinetics were used to update the distribution of sensitizer and reacted singlet oxygen. Oxygen depletion due to
photochemical consumption or vascular shutdown was also incorporated in the model. The adjoint method was
applied to calculate the PpIX fluorescence and 1270 nm singlet oxygen luminescence reaching the skin surface
in each time increment. The time-resolved evolution of the fluorescence and cumulative SOLD signals during
treatment were compared to the time-resolved volume-averaged distribution of reacted singlet oxygen in the
dermis layer for typical clinical PDT conditions. Approximate linear relationships were observed over most of
the treatment time.
Molecular mechanisms associated with ALA-PDT of brain tumor cells
Author(s):
Omar Alqawi;
Myrna Espiritu;
Gurmit Singh
Show Abstract
Previous studies have shown that low-dose PDT using 5-aminolevulinic acid (ALA)-induced photoporphyrin IX
(PpIX) can induce apoptosis in tumor cells without causing necrosis. In this study we investigated the molecular
mechanisms associated with apoptosis after ALA-PDT treatment in two brain glioma cell lines: human U87, and rat
CNS-1cells. We used high energy light at a short time (acute PDT) and low energy light at a long time of exposure
(metronomic PDT) to treat both cell lines. The cells were treated with 0.25 mM ALA at 5 joules for energy. We
found that CNS-1 cells were more resistant to ALA-PDT than U87 cells when treated by both acute and metronomic
PDT. To screen possible apoptosis mechanisms associated with acute and metronomic PDT, microarray analysis of
gene expression was performed on RNA from glioblastoma cells treated with either acute or metronomic ALA-PDT.
Within the set of genes that were negatively or positively regulated by both treatments are tumor necrosis
factor receptors. The expression of TNF receptors was investigated further by RT-PCR and western blotting. The
apoptosis mechanism of the cell death occurred through different pathways including BCL-2 and TNF receptors,
and in part caused by cleaving caspase 3. Interestingly, metronomic ALA-PDT inhibited the expression of LTβR
and the transcription factor NFκB. This inhibition was ALA concentration dependent at low concentrations.
Potentiation of ALA-PDT antitumor activity in mice using topical DMXAA
Author(s):
Allison Marrero;
Ulas Sunar;
Theresa Sands;
Allan Oseroff;
David Bellnier
Show Abstract
Photodynamic treatment of subcutaneously implanted Colon 26 tumors in BALB/c mice using the aminolevulinic acid
(ALA)-induced photosensitizer protoporphyrin IX (PpIX) was shown to be enhanced by the addition of the vascular
disrupting agent 5,6-Dimethylxanthenone-4-acetic-acid (DMXAA; Novartis ASA404). DMXAA increases vascular
permeability and decreases blood flow in both murine and human tumors. Sufficiently high parenteral DMXAA doses
can lead to tumor collapse and necrosis. We have previously reported marked enhancement of antitumor activity when
PDT, using either Photofrin or HPPH, is combined with low-dose intraperitoneal DMXAA. We now describe the first
attempt to combine topically-applied DMXAA with PDT. For this, DMXAA was applied two hours before PpIX-activating
light delivery. PDT with ALA-PDT alone (ALA 20%; 80 J/cm2 delivered at 75 mW/cm2) caused a 39%
decrease in tumor volume compared to unirradiated controls. Addition of topical DMXAA to ALA-PDT resulted in a
74% reduction in tumor volume. Diffuse correlation spectroscopy (DCS), a non-invasive blood flow imaging method, is
being used to understand the mechanism of this effect and to aid in the proper design of the therapy. For instance, our
most recent DCS data suggests that the 2-hour interval between the DMXAA and light applications may not be optimum.
This preliminary study suggests a potential role for topical DMXAA in combination with PDT for dermatologic tumors.
Generation of singlet oxygen and other radical species by quantum dot and carbon dot nanosensitizers
Author(s):
Roman Generalov;
Ingeborg Lie Christensen;
Wei Chen;
Ya-Ping Sun;
Solveig Kristensen;
Petras Juzenas
Show Abstract
Medicinal applications of luminescent semiconductor quantum dots are of growing interest. In spite of the fact that their
fabrication and imaging applications have been extensively investigated for the last decade, very little is documented on
photodynamic action of quantum dots. In this study we demonstrate generation of singlet oxygen and other radical
species upon exposure of quantum dots to blue light and therapeutic red light. Extent of radical production can be
readily modified by antioxidants. Lay and scientific communities are two sites concerning potential hazards and
enthusiastic applications of nanotechnology. Synthesis of quantum dots composed of less toxic materials is of great
interest. A new candidate is a ubiquitous element carbon, which on nanoscale exhibits strong photoluminescence.