Photo-therapy

K.O. Lenz

BMS Wilmington, DE

Photo-therapyOver the last few decades, photodynamic therapy has evolved to apromising new treating modality for cancer. The photo-sensitizers used,induce light sensitivity to a normal light insensitive chemical or physicalprocess. Present generation photosensitizers are derivatives of the bestof second generation photosensitizers introduced into or chemicallyattached to chemical devices. This modification is intended to increasethe biological specificity to deliver photosensitizers to a defined cell type.One of many such studies used Hypericin for photo-therapy of tumorcells via transferrin-conjugated PEG-liposomes. Does a targetingapproach work for photo sensitizers?

Photodynamic Therapy (PDT)

Photosensitizing agent is injected into the blood stream and absorbed by cells throughout the body

agent remains in cancer cells for a longer period of time than it does for non-cancerous cells

treated cancer cells are exposed to laser light at wavelength specific to photosensitizing agent

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

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.

Hypericin as a Photosensitizer Hypericin

naturally occurring

polycyclic aromatic quinone

binds mostly to cell membrane

photosensitizing agent

Mechanism Hypericin has been utilized in photodynamic therapy (PDT)

in the treatment of cancers as well as HIV

activated by visible light (400-700nm) reaction requires oxygen

upon photoactivation highly reactive singlet oxygen molecules are generated

quantum yield 0.73 causes irreversible cellular damage and tumor destruction reaction may be dose dependent

low dose = apoptosis (regulated) high = dose necrosis (unregulated-chemical)

Advantages of PDT

PDT offers advantages over traditional chemotherapy and radiation Minimal systemic toxicity highly selective not limited to skin cancer

can be utilized larger tumor masses

Cell & Tumor destruction Hypericin’s photodynamic actions include:

photohemolysis of RBC’s lipid peroxidation inhibition of

transcription factors growth factors MAP kinases

impairment of mitochondria function increases superoxide dismutase activity decrease cellular glutathione levels

These actions contribute the antiproliferative action

of Hypericin

Targeting via PEG-liposomes

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

Use of Transferrin in tumor Targeting

Tumors require high amounts of iron– cancer cells over express transferrin receptors

transferrin receptors have a rapid turnover transferrin is non-immunologic can be conjugated without losing biological activity

This makes transferrin a tumor seeking molecule

Liposomes

Conjugated liposomes need to be used since non-conjugated liposomes will be easily recognized and cleared by liver macrophages

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

PEG-liposomes are used to encapsulate and carry therapeutic agents

Combining PEG-Liposome and Transferrin

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

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 37C. Amount was determined by fluorescence measurement.

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.

Experimental DataIn vitro studies show that the photocytotoxicity and intercellular

accumulation of transferrin conjugated PEG-liposomes are somewhat higher than non-conjugated liposomes

Experimental conditions cellular accumulation

short incubation - slight increase long incubation (24hr) - more significant increase

Antiproliferative assay used to investigate phototoxicity of the three Hypericin

formulations Tf-Lip-Hyp had higher phototoxicity compared to Lip-Hyp Higher phototoxicity was seen between free Hyp and Tf-Lip-

Hyp during incubations under 4 hrs. Longer incubations show no significant differences

Experimental Data

Protein binding incubations containing FCS

Hypericin has a high affinity for albumin and lipoproteins in serum

substantial amount of Hypericin lost to albumin conjugated model unstable

Location of the Hypericin in the lipid bilayer of the liposome most likely plays an important role in

retention

Summary

Transferrin conjugated PEG-liposomes do increase the accumulation of hypericin in tumor cells

Hypericin is loosely bound to the liposome Hypericin has a high affinity for albumin 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

ConclusionThe 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.

Resources Research at the interface between chemistry and virology: development of a

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113-119 Parenteral Drug Delivery I. www.pharmj.com hypericin in cancer treatment: more light on the way. International Journal of

Biochemistry & Cell Biology, 2000, 34 221-241 Preparation and characterization of liposomes as therapeutic delivery systems:

a review. Pharmaceutica Acta Helvetiae 1995, 70 95-111