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LADEK ZDRÓJ

2003

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LASER SPECTROSCOPIC STUDY OF PHTHALOCYANINE DERIVATIVES SYNTHESIZED FOR PHOTODYNAMIC

THERAPY

András Grofcsik

Budapest University of Technology and Economics

Department of Physical Chemistry

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Photodynamic therapy (PDT)

Hematoporphyrin derivative (HpD)

Phthalocyanines (Pc)

Synthesis

Photophysical properties

in solutions

in vesicles

5-Amino-levulinic acid (ALA)

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Cancer therapies surgery radiotherapy chemotherapy

Photodynamic therapy (PDT): use of visible light in combination with a photosensitiser

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Number of publications in PDT

0

500

1000

1500

2000

2500

3000

1981-85 1986-90 1991-95 1996-2000

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Administration of

photosensitizer

Irradiation with visible light

Photosensitizer accumulates in

the tumour.

Tumour is selectively destroyed

Steps of photodynamic therapy

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Photodynamic effect:Cell destruction by

photosensitiser + visible light + O2

)()()( 110 TPSPSP ISCh

ProductsOesBiomolecul

OSPOTP

*2

1

*2

102

31 )()(

•TYPE II: energy transfer

•TYPE I: electron-transfer: Radicals and radical ions

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vessel

blood

The wavelength dependence of depth of penetration of light into soft tissue

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Requirements for the photosensitiser:

• Selective accumulation in malignant tissues• High absorbance between 600 and 800 nm• Chemical homogenity• Long triplet lifetime and sufficient triplet energy

(>94 kJ/mol)• Chemical, biological and photochemical stability• Little or no dark toxicity• Simple and cheap syntesis

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The first sensitiser used in clinical PDT:Hematoporphyrin derivative (HpD) Photofrin®

It is a mixture of compounds.

11 Hematoporphyrin

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HPLC analysis of HpD

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• Selective accumulation in malignant tissues• High absorbance between 600 and 800 nm• Chemical homogenity• Long triplet lifetime and sufficient triplet energy

(>94 kJ/mol)• Chemical, biological and photochemical stability• Little or no dark toxicity• Simple and cheap syntesis

Requirements for the photosensitiser:

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”Second generation" photosensitisers:

porphyrins

chlorins

bacteriochlorins

phthalocyanines (Pc)

naphthalocyanines

5-Aminolevulinic acid (ALA)

15Phthalocyanine

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00,20,40,60,8

11,21,41,61,8

500 550 600 650 700 750

(nm)

Ab

sorb

an

ce

b

a

Absorpion spectrum of a porphyrin (a) and a phthalocyanine (b) derivative (Ethanol solutions,

c = 1.5*10-5 mol dm-3)

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Synthesis

4 M = 2H

M = Zn

ROH, DMF, K2CO3

60 W, 20 min

3

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60 W, 3 minDBU

Zn(OAc)2

60 W, 15 minK2CO3

ROH, DMF CN

CNRO

CN

CNNO2

OR

RO

OR

N N

N

NN N

N

N

OR

M

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N

NN

M

NN

Phthalocyanine derivatives

I: M: Zn R: 4-tert-Bu-Ph-

II: M: Zn R: CH3O(CH2)2O(CH2)2-

III: M: Zn R: (2,6-dimethyl-4- N,N-dimethylamino- methylen)-phenyl-

IV: M: H2

R: CH3O(CH2)2O(CH2)2-

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Structure of III

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Experimental setup for studying triplet states

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Triplet lifetime and triplet absorption spectrum

II

IlgA

0

0

tOkAlnAln 20 400 450 500 550 600

0,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

Ethanol solution of I

(7,7*10--6 mol/dm

3)

Abs

orba

nce

[nm]

0 500 1000 1500 2000 2500

-0,03

-0,02

-0,01

0,00

0,01

0,02 Triplet decay

Abs

orba

nce

t [ns]

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Reaction of triplet Pc with molecular oxygen:

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023

1 )()( OSPcOTPc

The rate constant can be determined from the decay curves.

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0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,80

5

10

15

20

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I 0 [m

V]

Laser energy [mJ]

Ref I II IIIIV

Quantum yield of singlet oxygen formation

refref

phtalphtal m

m

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Triplet lifetimes (), second order rate constants and quantum yields of singlet oxygen formation

Pc (solvent) I (Et) I (To) II (Et) II (To) III (Et) IV (To)

(ns) 306 287 381 263 231 504

k*10-9

(M-1s-1)1.55 1.67 1.25 1.81 2.09 0.95

0.47 0.58 0.42 0.44 0.59 0.19

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Photosensitisers in vesicles

Vesicles are simple models of cell membranes.

DPPC (dipalmitoylphosphatidylcholine)

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27

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Temperature dependence of the rate constant (III in DPPC vesicles)

RT

EexpAk a

Arrhenius plot: Ea = 60.7 kJ

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20,4

20,8

21,2

21,6

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0,00300 0,00305 0,00310 0,00315 0,00320 0,00325 0,00330 0,00335 0,00340 0,00345

1/T [1/K]

ln k

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Use of 5-amino-levulinic acid (ALA) for PDT

COOH

CH2

CH2

C

O

CH2 NH2

ALA stimulates the cellular synthesis of an endogenous photosensitiser: Protoporphyrin IX

Administration of exogenious ALA causes the build-up of phototoxic levels of Protoporphyrin IX

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Advantages (over HpD):

•Treatment follows 2-4 hours after administration

•Systemic clearence of photosensitiser within 24 hours

•Treatment can be repeated within two days

•ALA can be administered topically

The method was approved by FDA in the 90s) In Hungary clinical trials started in 2001 (National Medical Center)

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Basal cell carcinoma - before treatment

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After PDT with ALA

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PARTICIPANTS Miklós Kubinyi

István Bitter

Viktor CsokaiJanka Tatai Klára Szegletes

Éva BacskayJános Brátán

Tamás VidóczyPéter BaranyaiLajos Csokonai Vitéz

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The End