Immune modulation using transdermal photodynamic therapy


J.G. Levy, R. Chowdhary, L. Ratkay, D. Waterfield, M. Obochi,

S. Leong, D. Hunt and A. Chan


Quadra Logic Technologies Inc.

520 West 6th Avenue

Vancouver, British Columbia, V5Z 4H5, Canada


(LR, DW, Faculty of Dentistry, University of B.C.,

Vancouver, B.C., Canada)


ABSTRACT


The photosensitizer benzoporphyrin derivative monoacid ring A (Verteporfin49 or BPD) has maximum absorption 
characteristics (690 nm) and biodistribution characteristics which permit activation of the drug in capillaries of the 
skin without causing skin photosensitivity (transdermal PDT). This permits targeting of cells in the circulation for 
selective ablation. Since BPD has been shown to accumulate preferentially in activated lymphocytes and monocytes, studies have been undertaken to determine the effect of transdermal PDT on murine models for 
rheumatoid arthritis (the MRL/lpr adjuvant enhanced model) and multiple sclerosis (the experimental allergic 
encephalomyelitis (EAE) model in PL mice). Localized transdermal PDT with BPD was found to be completely 
successful in preventing the development of adjuvant enhanced arthritis in the MRL/lpr mouse as well as 
improving the underlying arthritic condition of these animals. In the EAE model, in which an adoptive transfer 
system was used, it was found that transdermal PDT of recipients was effective in preventing EAE if treatments 
were implemented up to 24 hours after cell transfer but was not effective if given later, indicating the requirement 
for circulating T cells for effective treatment.


2. INTRODUCTION Benzoporphyrin derivative monoacid ring A (BPD) is a photosensitizes currently in clinical trials for cutaneous malignant lesions, psoriasis and for purging of leukemic bone marrows for autologous bone marrow 
transplantation. This photosensitizes has a maximum absorption peak at 690 nm, allowing significant tissue 
penetration and minimal attenuation by oxyhemoglobin (Fig. 1). The possibility that BPD in circulating blood 
could be activated by 690 nm light without causing skin photosensitivity was investigated in our laboratory, on the 
basis that early treatment following intravenous injection of BPD could target the drug in the circulation before it 
had distributed to the skin. Since BPD photobleaches readily upon exposure to light, this characteristic was used to 
determine the efficacy of transdermal treatment. These experiments demonstrated that mice give 1.0 mg/kg of 
BPD i.v. could withstand sufficient transdermal light treatment to effect about 80% photobleaching without 
sustaining skin photosensitivity or any measurable adverse reactions (liver enzymes, immune function, etc.).' 2


Further experiments with blood from HIV infected patients showed that treatment of blood with BPD and light 
could effect significant reductions in culturable virus, while causing no detectable hemolysis or reduction in 
leukocytes. Further analysis of these data using double labelling flow cytometry revealed that cells expressing the interleukin 2 receptor (IL-2R) were being selectively targeted.3 4 This observation that cells bearing activation markers were susceptible to this form of PDT led us to investigate the possibility that those autoimmune diseases, in which activated monocytes or T lymphocytes are known to be the cells responsible for the underlying pathology, might be amenable to transdermal PDT. The following reports on data obtained using two autoimmune disease models; the adjuvant enhanced rheumatoid arthritis model in the MRL/lpr mouse and the EAE (experimental allergic encephalomyelitis) model in the PL mouse.


3. METlIODS & RESULTS


3.1 Rheumatoid Arthritls (RA)


The MRL/lpr mouse is most widely studied as a model for lupus erythematosus. The animal suffers profound 
autoimmune disease characterized by lymphodenopathy, high levels of anti-DNA antibodies, dermatitis, 
underlying arthritis and ultimately immune complex disease and renal failure.5 6 Ratkay, Waterfield and 
co-workers recently demonstrated that a single injection of Complete Freund's Adjuvant (CFA) subcutaneously 
could induce an acute arthritis in the ankle joints of these animals within 30 days which, histologically, resembled 
human RA (7). This model was selected for study with transdermal PDT. Disease induction was followed by transdermal PDT to unshaved animals. Animals were injected with 1.0 mg/kg of BPD (liposomal formulation, 
Quadra Logic Technologies Inc.). Thirty minutes later, animals were placed in a light box as described previously7 
8 and exposed to 160 J/cm2 of broad spectrum red light (600-900 nm) over a ninety minute period. This treatment 
was performed immediately following adjuvant injection and 10 and 20 days following. All animals were 
sacrificed on day 30 and their ankles taken for histological evaluation. During the time course animals in the 
treatment group as well as CFA-enhanced and untreated controls, were monitored blind for evidence of disease. 
Results of these studies (Figs. 2 and 3 and Table 1) showed clearly that transdermal PDT delivered locally 
prevented development of the arthritic condition as well as improving joint condition in comparison to untreated 
controls with underlying disease (Fig. 3) since ankle width was actually reduced below that of control during the 
treatment period. This observation was supported by histological analysis in which PDT treated animals showed no 
bone deterioration during this time.


3.2 Experimental Allergic Encephalomyelitis (EAE)


EAE is considered to be a model for multiple sclerosis.9 In mice it is triggered by injection with myelin basic 
protein (MBP) and is mediated mainly by MBP specific T cells which attack the myelin sheath of the central 
nervous system, infiltrate the area, and cause progressive paralysis of the hind limbs.l A method was developed in 
our laboratories whereby MBP primed splenocytes or lymphocytes from PL mice could be cultured in vitro with MBP and IL-2 and transferred to naive recipients and cause disease in 100% of the recipients in an almost 
synchronous manner. This procedure was used to study transdermal PDT in this model, as depicted in (Fig. 4a, b). 
Initially a feasibility study was conducted, in which cells to be transferred were preincubated with 100 ng/ml of 
BPD for 60 minutes in the dark, washed and resuspended in 200 y1 of medium containing 5 % FCS. Fifty y1 of 40 yg/ml of BPD was then added to the cells, and the mixture was immediately injected i.v. into naive PL recipients. 
These animals were given transdermal PDT using a box containing narrow spectrum light emitting diodes (690 nm i: 10 nm) for 20 min (15 J/cm2) (Fig. 4a). Results (Fig. 5) showed that treated animals did not develop EAE whereas controls receiving untreated cells did. Further experiments were performed in which transdermal PDT was performed at 24, 48 and 120 hours following cell transfer (Fig. 4b). Results (Pigs. 6), show 
that transdermal PDT could effectively reduce or abrogate disease development if treatment was performed within 
24 hours following cell transfer but was not effective if given later. 


4. DISCUSSION AND CONCLUSIONS


4.1 Rheumatoid Arthritis


The transdermal PDT studies using the RA model of MRL/lpr mice have shown definitively that exposure of the 
hind limbs of mice to red light following i.v. administration of BPD prevented the development of arthritic 
symptoms in these animals. This model was utilized to develop principles for localized transdermal PDT since 
animals were unshaven and light was only applied from beneath. When plasma levels of BPD were determined in 
treated mice (immediately following treatment) in comparison to untreated animals, it was found that about 50% 
of the circulating BPD had been photobleached by the treatment. Therefore, it is unclear as to whether the effect 
seen resulted from treatment of cells within the ankle joint or cells within the circulation. Determination of this 
question requires further investigation. Nevertheless, the effect seen was profound and validates this approach, 
regardless of the location of targeted cells.


4.2 EAE


The preliminary work on the EAE model reported here again supports the hypothesis that activated T cells can be 
targeted by transdermal PDT, since the cell population adoptively transferred to treated recipients contains 
essentially only activated T cells. The observation that the transdermal PDT effect was lessened and eventually 
eliminated over time following cell transfer is not surprising since autoreactive cells introduced i.v. have been 
shown to rapidly "home" to their target, the myelin sheath where autoimmune damage might be initiated fairly rapidly.lt Regardless, this model is an interesting one in which to study the fate of transferred cells since they 
contain a prevalent T cell receptor phenotype (VCY4) which can be detected using the polymerase chain reaction 
(PCR).


In conclusion, these pilot studies have shown some promise in the potential for photosensitizers to effect immune 
modulation such that they might have application in the treatment of autoimmune disease.


5 REFERENTS


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