Non-oncologic potentials for photodynamic therapy

Anna M. Richter', Rubinah Chowdhary2, Leslie Ratkay3, Ashok K. Jain2,
Alice J. Canaan2, Howard Meadows2, Modestus Obochi2,
Douglas Waterfield3 & Julia Levy'

'Quadra Logic Technologies Inc., 520 West 6th Avenue,
Vancouver, B.C., VSZ 4H5, Canada

2University of British Columbia, Department of Microbiology
#300-6174 University Boulevard, Vancouver, B.C., V6T 1Z3, Canada

3University of British Columbia, Department of Oral Biology,
Faculty of Dentistry, Vancouver, B.C., V6T 1Z3, Canada

ABSTRACT

Benzoporphyrin derivative monoacid ring A (BPD is taken up rapidly (withill 30 minutes) by most cells. Rapidly 
dividing tumor cell lines and mitogen activated murine T lymphocytes were found to take up significantly more 
(5-10 fold) BPD than do normal splenic lymphocytes making them a potential PDT target. Experiments have shown 
that BPD can be activated in the blood of animals by whole body irradiation with red light, shortly after intravenous 
administration, in the absence of skin photosensitivity. During the treatment time, plasma levels of BPD were 
between 0.7 and 1.0 yg/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating 
BPD. When L1210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total 
body light treatment (160 J/cm2 of 590-900 nm light delivered over 90 min), significant reductions in circulating 
clonogenic tumor cells were observed in blood samples taken immediately following treatment. This "transdermal" 
treatment has been shown to be effective in preventing the development of hind limb arthritis in MRL/lpr mice.

2. INTRODUCTION

The main interest in photodynamic therapy (PDT) over the past decade has been focused on the potential of this 
technology in the treatment of solid tumors. The major first generation product, PHOTOFRINX has now received 
Board of Health approval in Canada, and regulatory approvals in Japan and some European jurisdictions are 
expected in the near term. A variety of other photosensitizers have been tested broadly in experimental animal 
tumor models, and a number are currently in clinical trials, mainly for the treatment of solid tumors. In addition to 
oncologic applications in PDT, a number of other clinical conditions may be candidates for PDT, based on a number 
of preclinical investigations. These include: skin disorders such as psoriasis, ophthalmic conditions like macular 
degeneration, atherosclerotic plaque and restenosis, bone marrow purging for treatment of leukemias with 
autologous bone marrow transplantation, and inactivation of viruses in blood or blood products. While these 
conditions appear disparate, common underlying pathological

characteristics provide a rationalization for the application of PDT. Many photosensitizers appear to have the 
common property that they concentrate somewhat selectively and rapidly in the endothelial cells comprising 
neovascular vessel walls. Activation by light under these conditions effects thrombus formation and vascular shut 
down of the neovasculature, resulting in a necrotic process of tissues being supplied by the vasculature. Since 
psoriasis, macular degeneration and atherosclerosis (via the vasa vasorum) all share the common pathology of 
neovascular involvement, it is not surprising that these conditions may be candidates for PDT. However, 
neovasculature is not a significant element in restenosis, bone marrow purging or viral inactivation; therefore, other 
physiological characteristics must be invoked to explain the PDT potential for this varied assortment of diseases. 
The present report will provide information which may help to explain a connection between these diverse 
conditions.

Benzoporphyrin derivative monoacid ring A (BPD) is a hydrophobic hematoporphyrin derived chlorin like 
photosensitizer which has shown promise in phase I/II clinical trials for the treatment of skin tumors and psoriasist 
2. Further, preclinical experiments have established that BPD and light can selectively eliminate leukemia 
progenitor cells in the marrow of patients with chronic myelogenous leukemia while sparing normal progenitors3. 
Studies on the ability of BPD and light to destroy indicator envelope viruses in whole human blood was established 
using both vesicular stomatis virus (VSV) and herpes simplex virus4. Further studies established that Sindbris but 
not EMCV (a nonenvelop virus) were also efficiently destroyed by BPD and light (Table 1). These early 
experiments led to investigation of the effects of BPD and light on the retroviruses, feline leukemia virus (FeLV) 
and human immunodeficiency virus (HIV), viruses which are normally found associated with activated lymphocytes 
as opposed to being free in blood. Results of our studies established that PDT on either FeLV or HIV infected blood 
samples resulted in extensive reductions in infectious virus in the absence of measurable hemolysis or leukocyte 
destruction5 6. Analysis of data has shown that BPD and light have selectivity for leukocytes which bear "activation 
markers" in blood. Since activated CD4+ cells are those in which HIV and FeLV replicate, the virucidal effect could 
be explained by the selectivity of this photosensitizer for these cells in blood.

Activated T cells are responsible for the symptoms of a majority of autoimmune conditions. The work reported here 
constitutes early results on studies which address questions regarding the possible mechanisms by which PDT can 
target activated T cells as well as the possibility that PDT could be used as a treatment for autoimmune conditions.

3. METHODS AND RESULTS

3.1. Photosensitizers

BPD (Fig. 1) was provided by Quadra Logic Technologies and was used as a liposomal formulation (Formulation 
was carried out by the Formulation Department of Lederle Laboratories). BPD has a maximum absorption peak at 
690 nm (Fig. 2), a wavelength which is above the absorption of hemoglobin. Therefore light at this wavelength is 
not significantly attenuated by blood or erythrocytes.

3.2. Uptake Studies

In order to determine whether activated T cells selectively took up BPD (thereby explaining the apparent selective 
sensitivity of this subpopulation) experiments were undertaken to study the uptake of BPD by normal mouse 
splenocytes compared to splenocytes from mice inoculated 24 h earlier with concanavalin A (Con A), a CD4+ T cell 
mitogen. Splenocytes were isolated and incubated with BPD (at 3,ug/mL in medium containing 10% FBS) for 30 
min. Cells were washed and cell content of BPD was assessed by fluorescence measurement as described 
previously7. Cell content of BPD was calculated on a per cell and a ,ug/mg protein basis. The results (Fig. 3) show 
that cells from mice stimulated with Con A take up approximately twice as much BPD as do splenocytes from 
control animals. Since only about 20% of splenocytes are activated by Con A, it was possible that the two fold 
difference in uptake was an under-estimate of differentials between activated and resting cells. Further experiments 
were carried out in vitro with mouse splenocytes cultured in the presence or absence of Con A. Cells were harvested 
following 24 and 48 hours, and incubated with BPD to determine cell uptake. The results (Fig. 4) confirmed that 
under these conditions the difference in uptake between activated and control cell populations was more 
pronounced. This is expected, since a majority of cells in culture with Con A would be activated. These data 
therefore confirmed that activated T cells do take up more BPD than their quiescent counterparts. This may account 
for the selective PDT effects seen.

3.3. Transdermal Activation BPD

Studies have been undertaken in our laboratory to determine whether a therapeutic window existed in which we 
could photoactivate BPD in the vasculature without causing skin photosensitivity; i.e., at a point following injection 
when plasma levels were in the range of 0.5 - l.5 ,ug/ml and the photosensitizer had not yet accumulated in the skin. 
Preliminary experiments have established that such a window exists. Mice were shaved and depilated, inoculated 
with 0.5 - 1.0 mg/kg BPD, kept in the dark for 60 minutes and subsequently exposed to whole body irradiation with 
broad spectrum red light (560-900 nm at 30 mW/cm2) for 90 minutes. Total light dose was 160 J/cm2 (as measured

using an IL 1350 photometer, International Light Inc.). Under these conditions, animals developed no skin 
photosensitivity during a two week observation period. However, when blood levels of active BPD were assessed 
following the 90 minute light exposure and compared to control animals, it was found that BPD levels, as 
determined by fluorescence, had dropped by 70-80% (Fig. 5, Table 2). It was concluded that the drop in active BPD 
following light exposure resulted from photoactivation and photobleaching of circulating BPD, thus indicating that 
activation of photosensitizers in the vasculature in the skin was possible, in the absence of skin photosensitization.

3.4. Pharmacological Model

A pharmacological model was subsequently tested to determine whether cells which had taken up high levels of 
BPD could be targetted by this transdermal procedure. Murine tumor cells (either P8 15 or L1210) were 
preincubated with BPD (at 100 ng/mL) following which they were injected i.v. into mice (2X106 cells in 0.2 ml of 
PBS together with 50 ,ul (egg) of additional BPD). Animals were immediately exposed to whole body red light for 
90 minutes. Animals were sacrificed immediately after treatment and blood levels of BPD and surviving tumor cells 
were determined and compared to untreated controls. Results are summarized in Table 3. It can be seen that this 
transdermal treatment effected both a reduction in the number of tumor cells and in the levels of plasma BPD in 
comparison to controls. It is of interest that the percentage of tumor cells killed correlates well with the amount of 
BPD photobleached, when individual experiments are compared (Fig. 6). These models provided evidence that the 
hypothesis that cells which take up high levels of the photosensitizer BPD can be

selectively targetted by transdermal PDT without causing skin photosensitivity.

Table 3. Results obtained in a pharmacologic model using Balb/c and DBA/2 mice and L1210 and P815 cells. 
Tumor cells (2 x 106/mouse) pre-loaded in vitro with BPD (100 ng/mL; 10% FBS), and BPD (2 yg/mouse) were 
injected intravenously immediately before the light treatment. Following the whole body exposure to red light (160 
J/cm2) the percent reduction of clonogenic tumor cells in blood and the percent of photodegraded BPD in blood 
were determined in relation to dark controls.

3.5. Autoimmune Model

MRL/lpr mice develop an autoimmune condition which resembles human systemic lupus erythematosus (SLE). 
These animals develop high levels of autoantibodies including anti-DNA antibodies and massive lymphadenopathy. 
This disease progresses and animals usually die at 6-7 months from a number of complications including renal 
failure. A finite percentage (about 5-10%) of them develop acute arthritic symptoms in their hind legs at the later 
stages of their disease. However, the incidence of acute arthritis is markedly enhanced (75-80%) if animals are 
injected intradermally with Freund's adjuvant containing Mycobacterium tuberculosis8. Using this model, MRL/lpr 
mice were injected with adjuvant and treated three times with BPD and light starting 4 h following adjuvant 
injection at 10 day intervals. The dosimetry involved i.v. injection of BPD (0.7 mg/kg), 60 minutes in the dark 
followed by 90 minutes of exposure to broad spectrum red light from beneath in such a manner that their feet and 
forepaws received the highest light exposure. The incidence of symptomatic arthritis was monitored in three groups 
of animals; those receiving adjuvant induction, those receiving adjuvant induction plus transdermal PDT at 10 day 
intervals, and those receiving no treatment. The incidence of arthritis as judged by erythema and swelling of the 
joints in the three groups is shown in Fig. 6. Histological evaluation of joint pathology confirmed the visual 
assessments and joint measurements (Fig. 7). Therefore, this model has established that transdermal activation of 
BPD had an immune modulatory effect on these animals which prevented the onset of adjuvant induced arthritis.