1. Photo-therapy K.O. Lenz BMS Wilmington, DE

2. Photo-therapy

3. Photodynamic Therapy (PDT) S Photosensitizing agent is injected into the blood stream and absorbed by cells throughout the body S agent remains in cancer cells for a longer period of time than it does for non-cancerous cells S treated cancer cells are exposed to laser light at wavelength specific to photosensitizing agent S agent absorbs light and produces active form of oxygen that destroys cancer cell  light exposure needs to be timed carefully so that it occurs when most of the photosensitizing agent has left the healthy cells but is still present in the cancer cells

4. Light Exposure How is light directed to cancer cells? Laser light used in PDT can be directed through a fiber optic. The fiber optic can be placed close to the cancer and deliver the proper amount of light. The fiber optic can be directed through devices such as a bronchoscope to treat lung cancers, or through a endoscope into the esophagus for treatment of esophageal cancer.

5. Hypericin as a Photosensitizer S Hypericin S naturally occurring S polycyclic aromatic quinone S binds mostly to cell membrane S photosensitizing agent

6. Mechanism Hypericin has been utilized in photodynamic therapy (PDT) in the treatment of cancers as well as HIV Sactivated by visible light (400-700nm) Sreaction requires oxygen S upon photoactivation highly reactive singlet oxygen molecules are generated S quantum yield 0.73 Scauses irreversible cellular damage and tumor destruction Sreaction may be dose dependent S low dose = apoptosis (regulated) S high = dose necrosis (unregulated-chemical)

7. Advantages of PDT S PDT offers advantages over traditional chemotherapy and radiation SMinimal systemic toxicity Shighly selective Snot limited to skin cancer S can be utilized larger tumor masses

8. Cell & Tumor destruction S Hypericin’s photodynamic actions include: Sphotohemolysis of RBC’s Slipid peroxidation Sinhibition of S transcription factors S growth factors S MAP kinases Simpairment of mitochondria function Sincreases superoxide dismutase activity Sdecrease cellular glutathione levels These actions contribute the antiproliferative action of Hypericin

9. Targeting via PEG-liposomes S Targeting –used to optimize the therapeutic effect by maximizing the accumulation of agent in the tumor tissue –minimize photo sensitivity of normal tissue Hypothesis: embedding hypericin in tumor targeted liposomes will increase the specific delivery of the compound to tumor cells, decreasing the accumulation in non-malignant tissue and reducing the photo-toxic effects of the skin

10. Use of Transferrin in tumor Targeting S Tumors require high amounts of iron –cancer cells over express transferrin receptors u transferrin receptors have a rapid turnover u transferrin is non-immunologic u can be conjugated without losing biological activity This makes transferrin a tumor seeking molecule

11. Liposomes S Conjugated liposomes need to be used since non-conjugated liposomes will be easily recognized and cleared by liver macrophages S PEG-ylated liposomes are often referred to as “stealth liposomes” due to their ability to remain in the blood for up to a hundred times longer than conventional liposomes S PEG-liposomes are used to encapsulate and carry therapeutic agents

12. Combining PEG-Liposome and Transferrin S Transferrin was conjugated to the distal end of the lipid conjugated PEG molecule so not to hinder the binding of the complex with the tumor cell receptor

13. Embedding Stability Stability of Hypericin embedded in non-conjugated PEG- liposomes. A 10uM suspension of Lip-Hyp was incubated for different time periods in presence of 10% FBS at 37 C. Amount was determined by fluorescence measurement.

14. Intracellular Accumulation Intracellular concentration of Hypericin in HeLa cells after incubation with free Hypericin (Hyp), Hypericin embedded in non-conjugated PEG-liposome (Lip-Hyp) and Hypericin embedded in Tf-conjugated PEG-liposome (Tf-Lip- Hyp) in presence of 10% FBS.

15. Experimental Data In vitro studies show that the photocytotoxicity and intercellular accumulation of transferrin conjugated PEG-liposomes are somewhat higher than non-conjugated liposomes S Experimental conditions Scellular accumulation S short incubation - slight increase S long incubation (24hr) - more significant increase SAntiproliferative assay S used to investigate phototoxicity of the three Hypericin formulations S Tf-Lip-Hyp had higher phototoxicity compared to Lip-Hyp S Higher phototoxicity was seen between free Hyp and Tf-Lip-Hyp during incubations under 4 hrs. Longer incubations show no significant differences

16. Experimental Data SProtein binding S incubations containing FCS SHypericin has a high affinity for albumin and lipoproteins in serum Ssubstantial amount of Hypericin lost to albumin Sconjugated model unstable Location of the Hypericin in the lipid bilayer of the liposome most likely plays an important role in retention

17. Summary S Transferrin conjugated PEG-liposomes do increase the accumulation of hypericin in tumor cells S Hypericin is loosely bound to the liposome S Hypericin has a high affinity for albumin S Passive release of hypericin from liposome to serum components prior to reaching tumor cells is a major issue A necessary condition for an effective tumor targeting drug delivery vehicle is stable drug encapsulation

18. Conclusion The unstable incorporation of hypericin in transferrin conjugated PEG-liposome makes this model unsuitable for in vivo testing. As can be seen placement of hypericin plays an important role its accumulation in tumor cells. Since hypericin is a lipophilic molecule it is poorly retained by the liposome.Therefore, for this method to be affective the hypericin needs to be embedded in a liposome containing several bilayers (multilamellar), which would increase the affinity of hypericin to liposome and decrease leakage to surrounding serum components. If the molecule was designed to overcome the unstable incorporation of hypericin it would be a interesting and potential model for cancer and HIV therapy.

19. Resources l Research at the interface between chemistry and virology: development of a molecular flashlight. Chem. Rev. 1996, 96, 523-535 l Transferrin-mediated targeting of hypericin embedded in sterically stabilized PEG-liposomes International Journal of Oncology 2002, 20: 181-187 l Hypericin in phototherapy. Journal of Photochemical and Photobiology 1996, 36 113-119 l Parenteral Drug Delivery I. www.pharmj.com l hypericin in cancer treatment: more light on the way. International Journal of Biochemistry & Cell Biology, 2000, 34 221-241 l Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharmaceutica Acta Helvetiae 1995, 70 95-111