Biophysical, in vitro, ex vivo, and in vivo evidence for the efficacy of three novel Os(II) based photosensitizers in photodynamic therapy mediated by visible (400-750 nm) and NIR (810nm) wavelengths Pavel Kaspler1, Savo Lazic1, Yaxal Arenas1, Jamie Fong1, Kamola Kasimova1, Arkady Mandel1, and Lothar Lilge1,2 Theralase, Toronto, Ontario 2 University of Toronto, Department of Medical Biophysics, Toronto, Ontario 1 Abstract We describe the biophysical, in vitro, ex vivo, and in vivo characterization of three novel photodynamic compounds (PDCs) based on a central Os(II) core: OsH2IP, OsH2B, and OsH2dppn. These PDCs absorb light between 400 and 800nm and are resistant to photo bleaching from green, red, and near infrared light. In vitro experiments in human glioma (U87) and transitional carcinoma (HT1376) cell lines showed the PDCs to be highly cytotoxic under a variety of wavelength exposures. Dark toxicity was found to be low for our PDCs ranging from 179 to 395 uM. OsH2dppn showed a high therapeutic ratio (Dark LD50 / PDT LD50) in green (3555, U87), red (63.9, HT1376), and near infrared (12.0, HT1376) light. OsH2IP had a lower therapeutic ratio in red (7.3, HT1376) light compared to OsH2dppn (p<0.01), but showed no significant difference from OsH2dppn in near infrared light (8.1, HT1376). H2B, while having low dark toxicity (LD50=395 uM) and moderate therapeutic ratio (22.6 in green, 4.9 in red, and 5.2 in near infrared), demonstrated a PDT effect in hypoxia suggesting that Os-based PSs are able to switch from a Type II to a Type I photo-effect. OsH2IP can attach to the rat urothelium after a short incubation, highlighting its potential use in bladder cancer therapy. In vivo growth delay studies were realized for OsH2IP. Subcutaneous heterotopic tumors were induced by injecting colon adenocarcinoma CT26.CL25 cells into the mouse hind leg. 5x5 mm tumors were injected intratumorally with 3mg/kg of OsH2IP, followed by 808 nm PDT treatment at 300mW/cm2 for a total energy of 600J/cm2. We observed consistent tumor regression following PDT treatment. Tumor regression is a minimum requirement for tumor protection, as reinjection of CT26.CL25 cells into the opposite flank resulted in no tumor formation. These findings demonstrate that NIR PDT leads not only to longstanding clearance of CT26.CL25 tumors, but also could provide long-lasting protection against further tumor cell re-challenges in young (8-10 weeks) and aged (12-14 months) mice. In summary, we developed three novel Os(II) based photosensitizers that absorb in a wide range of wavelengths, show strong light mediated cytotoxicity in human tissue culture systems, are able to cause complete remission in in vivo tumor models, and appear to be able to induce a potential immunemediated tumor protection response. The strong potency and efficacy of the PDCs in the phototherapeutic window (530−850 nm) will allow for the optimization of treatment based on the unique pathophysiological status of the patient and the clinical stage of the underlining medical condition, making them attractive clinical candidates for advanced anticancer PDT. Materials and Methods Initial Tumor injection-9 Days 0 Months 10 90 21 22 Aged matched controls Cells plating 15,000 cells/well Photosensitizer loading 6 hours incubation Photosensitizer removal (replaced by pyruvate-free medium) U87: DMEM CT26.WT: RPMI 1640 F98: DMEM 2nd injection 30 PDT 21ST day tumor N N Chemical structures of photosensitizers Incubation in dark 21 hours Dark Irradiation depending on the light source: 1) 530 nm 2) 635 nm 3) 808 nm In vitro approach for measuring LD50 of photosensitizers Terminology End point due to tumor size Natural end point due to old age Old aged deterioration 1 YEAR TUMOR FREE Y Y Viability staining Presto Blue, 1 hour (Phenol Red-free medium) Reading fluorescence 560/600 nm 3rd injection PDT CT26 Cl25 tumor injection 2.5e5 cell in 100μl, in dorsal area over 30 sec With a tumor size of 56mm, 100 μl/20g of 3mg/kg of PS, 4 hour incubation and 600 J/cm2 808nm In vivo PDT approach to acertain long term immunological memory Os(II) Photosensitizer Absorption OD Ratio Results 0.12 0.1 1 OsH2IP Bleaching 0.5 0 5E+20 Photon 0.08 808nm OD 0.06 OD Ratio 0.04 0.02 0 400 500 600 Wavelength(nm) OsH2B 700 Os2IP 1 635nm 0.5 5E+20 Photon OsH2dppn 808nm Photosensitizer absorption between 400 and 800nm. All three photosensitizers absorb at green (525nm), red (635nm), and near infrared (800nm) wavelengths. 525nm OsH2B Bleaching 0 800 1E+21 Exposure/cm2 1E+21 Exposure/cm2 635nm 525nm OsH2IP and H2B are resistant to photobleaching. The peak absorbance (350nm) was picked to calculate the OD ratio between bleached sample and unbleached sample. OsH2IP attached to intact (left) and damaged (right) urothelium after brief local application to rat bladder. 2mM OsH2IP solution was applied to the rat bladder with and without urothelium and incubated for 30 minutes. Bladders were washed with water to remove nonattached OsH2IP. Bladders were immediately snap frozen and sectioned. Wavelength (nm) 0.14 OsH2IP 0.2 Water OsH2dppn 0.1 OsH2B Urine 0.05 OD 0.1 OD 0.07 OD 0.15 0.05 0 400 500 600 700 800 500 Wavelength (nm) Water 0 0 400 600 700 800 400 500 Water 700 800 Wavelength (nm) Wavelength (nm) Urine 600 Water Urine Urine OsH2B Red 5 (U87) NIR 5 (U87) OsH2dppn Green Red NIR 3555 (U87) 64 (HT1376) 12 (HT1376) Inverse therapeutic ratios show a strong PDT effect in green, red, and NIR light. The inverse therapeutic ratio was calculated by dividing the dark toxicity LD50 by the PDT LD50. ** *** *** ** * * * Photosensitizers are stable in human urine. Each photosensitizer was diluted to 10uM in human urine and incubated at room temperature for one hour. Absorbance measurements show that all photosensitizers continue to absorb between 400 and 800nm. Interestingly, urine incubation increased photosensitizer absorption relative to water incubation. Green Not tested OsH2IP Red NIR Green 7 (HT1376) 8 (HT1376) 22 (U87) PDT efficacy of Os-based OsH2B in normoxic and hypoxic conditions under irradiation by red (635 +/- 25 nm, 90 J/cm-2, 108 mW cm-2) light. Number of absorbed photons per cm-2 is indicated for each group. White bars denote dark toxicity (no light) and red bars PDT effect (total cell kill with light alone and PS alone cell kills subtracted). The values are expressed as percent of control (no light, no PS). Group size and averages with standard errors are shown. Significance of cell kill as above zero (by one-tailed T-test): * – P<0.05, ** – P<0.01, *** – P<0.001. Discussion Outcome of OsH2IP PDT (3 mg kg-1 OsH2IP, 808 nm, 600 J cm-2) performed on 2-3 month old BALB/c mice with tumors induced by injection of CT26.CL25 cells (murine colon carcinoma, immunogenic subclone). Second injection of CT26.CL25 cells shows that succesful PDT treatment resulted in tumor resistance. Mice remained resistant for at least 12 months. In vitro cell kill studies in U87 glioma and HT1376 bladder human cell lines show a PDT effect in green, red, and NIR wavelengths for all photosensitizers tested. The ability to produce a consistent cytotoxic effect in response to light opens up many possibilities for the use of our photosensitizers in medical applications. OsH2IP is able to produce cell kill in the NIR range, allowing for the use of deep tissue penetrating NIR lasers to be used in conjunction with OsH2IP. This raises the possibility of using OsH2IP as a novel photosensitizer in the treatment of deep tissue cancers. All the photosensitizers are stable in human urine. We show that OsH2IP attached to intact and damaged rat urothelium after a brief local exposure, raising the possibility of utilizing OsH2IP for treatment of bladder cancer. Current experiments are testing if changes in incubation and washing parameters can enhance OsH2IP attachment to damaged urothelium. OsH2dppn has a very good therapeutic ratio in green light, making it a promising candidate for topical PDT applications, such as skin cancer treatment. Intriguingly, OsH2B shows PDT activity in hypoxic conditions, opening up avenues for its use as a treatment for bulky tumors with hypoxic cores. PDT using 3 mg kg-1 OsH2IP and near infrared light source (805 nm, 600 J cm-2) was able to destroy tumors induced in 2-3 month old BALB/c mice by CT26.CL25 cells (murine colon carcinoma, immunogenic subclone). Reinjection of CT26.CL25 cells 13-23 days post-PDT resulted in only weak and temporary tumor regrowth or no tumor regrowth suggesting at least short-term tumor rejection immunogenic response. Reinjection of CT26.CL25 cells 10-11 months post-PDT also did not result in any tumor regrowth (more than 3 months follow up) suggesting persistence of long-term tumor rejection immunogenic response. The ability of CT26.CL25 cells to induce tumors in 10-11 month old mice was verified in age-matched control animals that demonstrated considerable tumor growth (in 3 of 5 animals) approximately 1 month post-injection. The tumors were deep and diverse in the untreated animals and completely destroyed in the PDT treated animals, suggesting that OsH2IP PDT elicits a strong immune-mediated response. These findings could propel OsH2IP as a first line treatment for cancer if these results are replicated in humans. Acknowledgements and Funding