Photodynamic Therapy

Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

Photodynamic Therapy (PDT)

• Origin of tumors and the principles of their treatment

• Principles and history of PDT

• Photo-physical and -chemical aspects

• Photosensitizers (PS) of 1. generation

• Endogenous photosensitizer

• Photosensitizers of 2. and 3. generation

• Summary

Origin of tumors

A B

C

A. Individual cells with modified genome

B. Hyperplasia: mutated cells are phenotypically identical with the healthy ones but they multiply faster

C. Dysplasia: abnormalities in cell shape and orientation

D

E

D. Noninvasive carcinoma: The cells differ more in appearance and multiplication rate. The tumor does not spread to other tissues

E. Invasive carcinoma: Spreading out of the tissue of origin: individual cells are transported by cardiovascular and lymphatic system; a malignant tumor can lead to metastasis over the whole body

Origin of tumors

Principles of tumor treatment

Traditional• Surgery• Radiotherapy• Chemotherapy

New developments• Boron neutron capture

therapy• Monoclonal antibody

therapy• Antigene or antisense

therapy• Photodynamic therapy

(PDT)

Principles of photodynamic therapy of tumors (PDT)

• Photosensitizers (PS) are not toxic “at dark”

• PS accumulate in tumors

• Illumination of the tumor leads to a) Fluorescence: diagnosis of the tumor

b) Killing of tumor cells

(Apoptosis, Necrosis) - PDT

History of photodynamic therapy of tumors

• 1900 Acridin exhibits photo-toxicity (Raab)

• 1903 Eosin applied against skin cancer (von Tappeiner)

• 1908 Photo-toxicity of porphyrins (Hausmann)

• 1913 Mayer-Betz tested photodynamic therapy with porphyrins on his skin

• 1924 Porphyrin enriched tissue exhibits red fluorescence upon illumination with UV radiation (Policard)

• 1942 Different retention of porphyrins in helathy and malignant tissues (Auler, Banzer)

• 1948 Diagnosis and treatment of cancer by hematoporphyrin and its complexes with zinc (Figge)

• 1961 Lipson developed a hematoporphyrin derivative (HpD).

• 1966 first successful breast cancer treatment by HpD (Lipson)

• 1978 first systematic clinical studies (Dougherty)

• Today a few thousand patients treated by HpD

Photosensitizers of the first generation

History of photodynamic therapy of tumors

Photosensitizers of the first generation

are oligomers (HpD) of Hematoporphyrin (Hp)

• Absorption at 405, 505, 525, 565 and 630 nm

• Emission at 635 and 700 nm• Accumulation in tumors

Hp

Dihemato-porphyrin- ether

Photophysics (Jablonski)

S1

Nonradiative transitions: knrT

knrS

Fluorescence: kf

Intersystem Crossing: kisc

Energy of the states

Phosphorescence: kp

Excited state reactions of photosensitizer in T1 state represent an additional nonradiative decay pathway. Reaction with O2 gives rise to singlet oxygen 1O2. Electron transfer reactions give rise to free radicals

Quantum Yields: F: f = kf / (kf + knrS + kisc)

ISC: isc = kisc / (kf + knrS + kisc)

P: p = isc kp / (kp + knrT)

What is 1O2?

• O2 is paramagnetic (in triplet

state) in the ground state (according to Hund rule)

• Because of spin restriction triplet oxygen 3O2 participates

only in non-selective radical reactions

E

95 KJ/mol

3O2 1O2

Electron configuration of 3O2

1269 nm

• Singlet oxygen 1O2 is very reactive and selective

Photochemistry

S1

O2

REAKCE Volné radikály

Energy of the states

(1O2 ) = isc k [3O2 ] / (k [3O2 ] + kp + knrT)

Type 2 reaction (energy transfer)

FREE RADICALS

Type 1 reaction (electron transfer) + O2

Reactive Oxygen Species (ROS):

Superoxide ·O2-

Hydroxyl rad. ·OH…

k

HpD accumulates preferentially in

membranes

Plasma membrane

Mitochondria

outer membrane

Nuclear membrane

inner membrane

Lysosomes

Endoplasmatic reticulum

Affected sites

Reactionsof 1O2 and ROS

with biomolecules

Peroxidation

Addition on cycles

Oxidation

Cause oxidative damage, which can lead ultimately to cell death

AFTERBEFORE

BEFORE – The photodynamic diagnostics (PDD) of a tumor

AFTER – The tumor tissue has been removed by PDT

Pros and cons of the 1. generation of photosensitizers

• Photophysics: high isc and (1O2 ), but relatively short wavelength absorption with a low absorption coefficient

• in vivo activity: low dark activity, high photodynamic activity, but relatively low selectivity of absorption in tumors

An example of HpD-PDT

INJECTION

intravenous

ACUMULATION IN TISSUE

ELIMINATION FROM

ACUMULATION IN TUMOR

TUMOR ILLUMANTION

weeks

NECROSIS

Healthy tissue Skin Serum

APOPTOSIS

• Photosesitizer protoporphyrin IX (Pp IX) is an intermediate of heme synthesis

• The physiological concentration of Pp IX is low because of a controlled expression of its precursor 5-aminolevulinic acid (ALA)

Endogenous PS:

Cells produce their own PS

Heme

ALAPp IX

COO-

CH2

CH2

C O

CH2 NH2

Ferrochelatase

MIT

OC

HO

ND

RIA

succinyl-CoA+

glycine

ALA-synthase

• The expression of ALA is feedback controlled via heme concentration

• Administration of exogenous ALA breaks the feedback control and results in accumulation of Pp IX

Endogenous PS:

Cells produce their own PS

Heme

ALAPp IX

COO-

CH2

CH2

C O

CH2 NH2

Ferrochelatase

MIT

OC

HO

ND

RIA

succinyl-CoA+

glycine

ALA-synthase

• Concentration of Pp IX is higher in cancer cells due to their higher metabolic activity and in some cases also due to decreased efficiency of ferrochelatase and increased efficiency of Pp IX synthesis from ALA

Photosensitizers of 2. generation:

- long wavelength absorption with large extinction coefficient- selective accumulation in tumor

Naphtalocyanine: ex = 820 nm

Porphycen:ex = 710 nm

Phtalocyanine:maximal ex = 740 nm

Chlorin e6: ex = 750 nm

Photosensitizers of 3. generation (selective acculmulation)

• Monoclonal antibodies bind selectively to an antigen on cancer cells

• The spacer is either cyclodextrin or Avidin-Biotin-system

PS

Spacer

Antibody

„Drug Targeting“

Summary

Limitations:Low penetration depth in tissue (ideal for skin cancer or with endoscopic illumination)

Advantages:Low costRelatively low side effects

Goals:High selectivity for cancer cellsOptimal illumination dose

Triplet Oxygen

Singlet Oxygen

Free radicals

REAKCE

Energy of states

Type II reaction

Type I reaction

Methodological outlook:Multiphoton excitation

• High intensity of ps- or fs-lasers• Excitation by light of double or

triple wavelength compared to single photon excitation

• Light of longer wavelength penetrates deeper to the tissue

Longwave excitation of a PS with shortwave absorption

Acknowledgement

The course was inspired by courses of:

Prof. David M. Jameson, Ph.D.

Prof. RNDr. Jaromír Plášek, Csc.

Prof. William Reusch

Financial support from the grant:

FRVŠ 33/119970