Vol. 186, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1992
Pages 1032-l
036
EFFECT OF PHOTOSENSITIZERS IN CHEMICAL AND BIOLOGICAL PROCESSES: THE MT0 MECHANISM IN PHOTODYNAMIC THERAPY Dezsti Central
Research H-1525
Institute Academy Budapest,
Gal
for Chemistry of Sciences P-0. Box 17,
of
the
Hungarian
Hungary
Received June 16, 1992
It is suggested that in the course of the application of photosensitizers irradiated for cancer therapy a real competition occurs between the singlet oxygen mediated effects and the interactions between the triplet photosensitizers and doublet radicals formed in tumor cells while processes between photosensitizer radicals and molecules are negligible. This hypothesis is strongly supported by experimental findings and reveals a novel approach for the synthesis of new photosensitizers. 0 1992
Academic
Press,
Inc.
In the last decade enhanced attention has been paid to the application of photosensitizers in chemistry, biology and medicine due mainly to the fast development of coherent optical light sources and of laser technique combined with fiber optics. Interestingly one of the most promising feature comes from the practical medical application called Photodynamic Therapy (PDT) according to which if certain photosensitizers such as haematoporphyrin (HP), its derivates (Hpd), phthalocyanines, etc., are administered to patients with malignant tissues in amounts 5-15 mg/kg followed by local irradiation of the cancer cells the destruction of the latter takes place 11-63. In spite of the fact that about 7-8000 patients have been treated, so far, in Phase II and Phase III clinical trials and the FDA approval is in due course in the USA for lung, bladder and superficial endobronchial cancer, the chemical mechanism of the overall procedure is far from well known [71. According to literature sources two main mechanisms are assumed in order to explain the chemistry of PDT C8-121. TYPE ONE (TO) mechanism assumes that irradiated Hp (or Hpd) yields excited singlet state molecules transformed into 0006-29 I X/92 $4.00 Cop.vright 0 I992 by Academic Press, Inc. All rights of reproduction in arty form reserved.
1032
Vol.
186,
No.
triplet ions,
BIOCHEMICAL
2, 1992
species. reacting as
BIOPHYSICAL
These latter result with stable molecules
TYPE TWO (TT) oxygen
AND
mechanism
destroying
is
agent
COMMUNICATIONS
in Hp radicals of the living
based
which
RESEARCH
on the
is
formed
or radicalcells.
effect
of
singlet
by an energy
trans-
fer between triplet Hp and triplet oxygen present in ganisms. Singlet oxygen is very active towards stable to
Although there in the literature
anism of
and
indicating
one of
the
with
respect
been
published.
the
two to
Kinetic suggest
are several supporting
the
and
further
observed
with to
The ing
the
the
of
isms
could
be given
kinetic
analysis
mechanisms
has
the
explain
called
several
details
approach
various
steps
not
results
TO mechanism,
realistic
the
the
that
active
with
resulting
species
photosensitizers in
the
leading
react
biological to
efficiencies
the of
using experimentel data. In first approximation the can
occurrence
experimental
a more
assumes
etc.)
compare
them,
of
between
independently ions,
of
the
actual
in
cells. of
formed radical us
to
competition
irradiation
Let
mechanism
lead
MT0 mechanism
species cals,
ism
PDT and
the
recent
development
TYPE ONE (MTO)
destruction
both
results referred TO- or the TT-mech-
of between
considerations
that
respect
or
competition
MODIFIED
the
possibility
mechanisms the
experimental either the
living ormolecules.
overall
rate
with
dur-
active
systems
effect the
formed
(radi-
observed
MT0 and for
the
in
PDT.
TT mechanMT0 mechan-
as
WMT0 = kMTO CHplxCRad'l where formed
[HplX and CRad'l by irradiation
gical
cells,
Laser experiments interaction negligible and doublet Consequently 3 Hp + *Rad'
mean the of Hp and
concentrations of active radicals present in the
species biolo-
respectively. flash photolysis performed in between while
excited
as well as single our Laboratory have singlet
interactions
radicals have for comparison reaction
between been of
of
triplet
Hp and
radicals
photosensitizers
thoroughly investigated the MT0 and TT mechanisms
was considered
WMTO
state
photon counting shown Cl31 that
exclusively,
- kMTO C3Hp1C2Rad'l
1033
that
C14-161. the is
is
BIOCHEMICAL
Vol. 186, No. 2, 1992
AND BIOPHYSICAL
molecules /-
Rv
3
Hp-lHpX-Hp
molecules Hp' p
TO mechanism
molecules
TT mechanism
---+O2
\
RESEARCCI COMMUNICATIONS
\ '(radicals)' Fig.
1.
Chemical pathways of photosensitizers
The various pathways represented in Fig. 1. The overall
rate
where CM011 means the singlet oxygen. that
'02
is
state
the
so far,
are
schematically
TT mechanism can be expressed
as:
11021LMoll
= kTT
'TT
A steady
suggested so far for the effect irradiated in malignant tissues.
assumed,
of
*MT0 mechanism
(3)
concentration
treatment
of molecules
for
consumed in its
singlet
interaction
reacting
oxygen, with
if
with
assuming
molecules,
gives:
dC102 I kl
dt" where
kl
tween
triplet
refers
to not
destructing
ing , simple substitution
the
rate
constant
Hp and triplet
Naturally, cell
C3HplC021 - k TT C1,21[Mo11
all
energy
TT mechanism,
of
that
the
into
oxygen loss
transfer,
(4)
From equations
is
results
(11,
= kl
transfer
be-
are efficient processes
considerable, in
the maximal
(quench-
therefore rate
of
the
(2)
According to in malignant
literature cells is
(5)
[3H~11021
and
(5)
kMTO CRad'l
(6)
co21
(WTT)max = kl
1 -l
molecules
by secondary
etc.) (3)
W MT0
mol
energy
is:
(WTTlmax
[02],
the
(4)
Oz.
singlet
agents,
of
= 0
the concentration data, quite low being about
1171. 1034
of oxygen, (1-5)~10-~
Vol.
186,
No.
With tions 201. tory
respect
it
exceed
BIOCHEMICAL
2, 1992
has 4-5
to
been
the
oxy
established
times
the
has
revealed of
1-1,
thus
CRad'l
that
it
is
in
in
the
Consequently
in
same such
while
k MTO is
from a kinetic tain
type
in the
of
the
quence.
view
Namely,
kl-
tuspin
as
mol
1021.
(2-6)x10'
rate
M-I s -l
constant
rate
c221
[15].
can be strongly
and even
kinetic
in PDT, it
the
photosensitizers.
basic
competitive
determining
activation
feature
tendency they
at
are
at higher
accumulation
of
under
cer-
MT0 mechanism to be studied
the
has an important
as a search
So far,
increased
C18-
transplanted
(0.5-1.5)~10-~
the actual rate of the 3 2 the HP + Rad' interaction
PDT can be formulated
(iii)
sources
interesting
mechanisms
(i)
tissues
future.
various
tions:
in
might
in our Laboratemperature)
magnitude
controlled
of
in
normal
However,
depends on the Beyond
stages of
MT0 mechanism
point
conditions.
in detail
literature
the
in
recently nitrogen
range
order
tissues
k m- MT0 kl
a diffusion
Therefore,
cancer
concentra-
cases:
(WTT)max
to
the
COMMUNICATIONS
radical
values
early
in
'MT0
According
in
performed (at liquid
the
varies
RESEARCH
peroxy
corresponding
C2.11 that
mice
BIOPHYSICAL
and/or
Kinetic ESR spectroscopy in frozen tissue samples
mors
of
AND
practical
present for
in the
novel
tailored
wavelengths;
of
the
consedevelopment
and more efficient
to
in malignant
role
following (ii)
indica-
low toxicity;
tissues;
(iv)
optimal
photobleaching. If sidered tizers
the
MT0 mechanism will
likely
to
play
to be used in
is expected cluding
to
react
a probable
extending
the
be strongly
a role,
a new requirement PDT can be introduced: its as an efficient
decomposition
role
of
supported
radical during
and con-
for photosensiactivated form
"scavenger"
photoactivation
inand thus
Hp as a carrier.
REFERENCES 1.
D. Russell,
2.
Plenum, 1983, Y. Hayata, T. vative
T.
Dougherty: p. 160) Dougherty:
in Cancer
(Tokyo,
Adv.
Exp.
Med.
Biol.
(New York
Lasers and Haematoporphyrin Igaku-Shoin, 1984) 1035
Deri-
Vol.
186,
3.
4.
No.
2, 1992
G. Jori:
In
(New
York
Land,
T.
H.
6.
Abstracts.
van
dynamic
C.
Press.
T.G.
BIOPHYSICAL
Photoprocesses Eds:
G. Truscott,
den
RESEARCH
in
R.V.
1985,
pp.
Truscott:
Bergh: 3rd
COMMUNICATIONS
Biology
and Medicine
Bensasson,
G. Jori,
E.J.
349-355)
Inst.
Biennal
J.D.
J.
p.
183,
in
Radiat.
Britain,
Meeting
of
(Buffalo,
Spikes:
Emiliani,
490
Chemistry
Association
G. Jori, Smith,
9.
Plenum
AND
Biol.,
2,
421-
(1986)
5.
8.
Primary
R. Potter, 452
7.
BIOCHEMICAL
New York
the
pp.
430-439.
International
New York
Topics in Plenum,
M. Delmelle:
1986,
Photo-
1990)
Photodynamics 1984)
Photochem.
(Ed.:
Photobiol.,
K.C.
37,
487-
(1983)
G.R. cine
Buettner: Primary Photoprocesses (Eds: R.V. Bensasson, G. Jori,
Truscott, 10.
J.
Moan:
11.
R.
Bonnett,
New York
Plenum
Photochem.
pp.
Photobiol.,
C. Lambert,
Sinclair,
Press,
T.G.
Truscott:
D.G.
Whitten:
in E.J.
341-344,
2,
E.J.
Biology and MediLand and T.G. 1985)
445-449
Land,
P.A.
Photochem.
(1984)
Scourides,
R.S.
Photobiol.,
2,
l-8
(1983) 12.
M. Krieg,
13.
T.
Vidbczy,
14.
A.
Seret,
145-155 15.
A.
16.
H.
S.
T.
19.
21.
Obi:
J.
Durai,
P.K.
Das,
S. Raja
Chem.
(1967);
de Vorst:
22.
(1984)
publication
Kinichi
Phys.
V.S.
Lett.,
Mosyenko,
137, A.D.
J.
Photochem.,
Phys.
Chem.,
Mulay, (1967);
J.
Duchesne,
Phys.
Med.
L.N. N-1.
danov, N.N. Zoz: T. Shulykovskaya, R.W. Redmond, 523-529 (1988)
S.E.
G.L.
255-260 Bullach:
of Oxygen in Biological 1974, pp. 191-200, in
Kinetics of N.M. Emanuel: (Naukai Moscow, 1977, pp. C.M. Dettmer, D.H. Driscoll,
I.L. 743
235-252
38, 95,
(1991)
Hiratsuka,
See e.g.:
493 20.
under
25,
A. Van De Vorst:
Ohutsa,
Determination Nauka Dumka, 18.
Photochem.,
(1987)
9130-9134
17.
Zemzam:
E. Gaudin,
Kawai,
senden:
J.
20,
Mulay: J. Ekvtimishvili,
Natl.
Biofizika, L. Siimegi: Braslavsky:
1036
"Polarographic
Entities" Russian) Tumor
(Kiev, Processes
in Russian) Wallace: Nature,
R. Goutier,
Biol.,
W. Fes-
(1987) in
Experimental 340-344, J.D.
Hug,
R.
Marchal,
305-309
492A. Van
(1975)
Cancer Inst., L.P. Kayushin,
39, 735G.N. Bog-
29, 298-301 (1984) under publication Chem.
Phys.
Letters,
148,
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1992
Pages 1032-l
036
EFFECT OF PHOTOSENSITIZERS IN CHEMICAL AND BIOLOGICAL PROCESSES: THE MT0 MECHANISM IN PHOTODYNAMIC THERAPY Dezsti Central
Research H-1525
Institute Academy Budapest,
Gal
for Chemistry of Sciences P-0. Box 17,
of
the
Hungarian
Hungary
Received June 16, 1992
It is suggested that in the course of the application of photosensitizers irradiated for cancer therapy a real competition occurs between the singlet oxygen mediated effects and the interactions between the triplet photosensitizers and doublet radicals formed in tumor cells while processes between photosensitizer radicals and molecules are negligible. This hypothesis is strongly supported by experimental findings and reveals a novel approach for the synthesis of new photosensitizers. 0 1992
Academic
Press,
Inc.
In the last decade enhanced attention has been paid to the application of photosensitizers in chemistry, biology and medicine due mainly to the fast development of coherent optical light sources and of laser technique combined with fiber optics. Interestingly one of the most promising feature comes from the practical medical application called Photodynamic Therapy (PDT) according to which if certain photosensitizers such as haematoporphyrin (HP), its derivates (Hpd), phthalocyanines, etc., are administered to patients with malignant tissues in amounts 5-15 mg/kg followed by local irradiation of the cancer cells the destruction of the latter takes place 11-63. In spite of the fact that about 7-8000 patients have been treated, so far, in Phase II and Phase III clinical trials and the FDA approval is in due course in the USA for lung, bladder and superficial endobronchial cancer, the chemical mechanism of the overall procedure is far from well known [71. According to literature sources two main mechanisms are assumed in order to explain the chemistry of PDT C8-121. TYPE ONE (TO) mechanism assumes that irradiated Hp (or Hpd) yields excited singlet state molecules transformed into 0006-29 I X/92 $4.00 Cop.vright 0 I992 by Academic Press, Inc. All rights of reproduction in arty form reserved.
1032
Vol.
186,
No.
triplet ions,
BIOCHEMICAL
2, 1992
species. reacting as
BIOPHYSICAL
These latter result with stable molecules
TYPE TWO (TT) oxygen
AND
mechanism
destroying
is
agent
COMMUNICATIONS
in Hp radicals of the living
based
which
RESEARCH
on the
is
formed
or radicalcells.
effect
of
singlet
by an energy
trans-
fer between triplet Hp and triplet oxygen present in ganisms. Singlet oxygen is very active towards stable to
Although there in the literature
anism of
and
indicating
one of
the
with
respect
been
published.
the
two to
Kinetic suggest
are several supporting
the
and
further
observed
with to
The ing
the
the
of
isms
could
be given
kinetic
analysis
mechanisms
has
the
explain
called
several
details
approach
various
steps
not
results
TO mechanism,
realistic
the
the
that
active
with
resulting
species
photosensitizers in
the
leading
react
biological to
efficiencies
the of
using experimentel data. In first approximation the can
occurrence
experimental
a more
assumes
etc.)
compare
them,
of
between
independently ions,
of
the
actual
in
cells. of
formed radical us
to
competition
irradiation
Let
mechanism
lead
MT0 mechanism
species cals,
ism
PDT and
the
recent
development
TYPE ONE (MTO)
destruction
both
results referred TO- or the TT-mech-
of between
considerations
that
respect
or
competition
MODIFIED
the
possibility
mechanisms the
experimental either the
living ormolecules.
overall
rate
with
dur-
active
systems
effect the
formed
(radi-
observed
MT0 and for
the
in
PDT.
TT mechanMT0 mechan-
as
WMT0 = kMTO CHplxCRad'l where formed
[HplX and CRad'l by irradiation
gical
cells,
Laser experiments interaction negligible and doublet Consequently 3 Hp + *Rad'
mean the of Hp and
concentrations of active radicals present in the
species biolo-
respectively. flash photolysis performed in between while
excited
as well as single our Laboratory have singlet
interactions
radicals have for comparison reaction
between been of
of
triplet
Hp and
radicals
photosensitizers
thoroughly investigated the MT0 and TT mechanisms
was considered
WMTO
state
photon counting shown Cl31 that
exclusively,
- kMTO C3Hp1C2Rad'l
1033
that
C14-161. the is
is
BIOCHEMICAL
Vol. 186, No. 2, 1992
AND BIOPHYSICAL
molecules /-
Rv
3
Hp-lHpX-Hp
molecules Hp' p
TO mechanism
molecules
TT mechanism
---+O2
\
RESEARCCI COMMUNICATIONS
\ '(radicals)' Fig.
1.
Chemical pathways of photosensitizers
The various pathways represented in Fig. 1. The overall
rate
where CM011 means the singlet oxygen. that
'02
is
state
the
so far,
are
schematically
TT mechanism can be expressed
as:
11021LMoll
= kTT
'TT
A steady
suggested so far for the effect irradiated in malignant tissues.
assumed,
of
*MT0 mechanism
(3)
concentration
treatment
of molecules
for
consumed in its
singlet
interaction
reacting
oxygen, with
if
with
assuming
molecules,
gives:
dC102 I kl
dt" where
kl
tween
triplet
refers
to not
destructing
ing , simple substitution
the
rate
constant
Hp and triplet
Naturally, cell
C3HplC021 - k TT C1,21[Mo11
all
energy
TT mechanism,
of
that
the
into
oxygen loss
transfer,
(4)
From equations
is
results
(11,
= kl
transfer
be-
are efficient processes
considerable, in
the maximal
(quench-
therefore rate
of
the
(2)
According to in malignant
literature cells is
(5)
[3H~11021
and
(5)
kMTO CRad'l
(6)
co21
(WTT)max = kl
1 -l
molecules
by secondary
etc.) (3)
W MT0
mol
energy
is:
(WTTlmax
[02],
the
(4)
Oz.
singlet
agents,
of
= 0
the concentration data, quite low being about
1171. 1034
of oxygen, (1-5)~10-~
Vol.
186,
No.
With tions 201. tory
respect
it
exceed
BIOCHEMICAL
2, 1992
has 4-5
to
been
the
oxy
established
times
the
has
revealed of
1-1,
thus
CRad'l
that
it
is
in
in
the
Consequently
in
same such
while
k MTO is
from a kinetic tain
type
in the
of
the
quence.
view
Namely,
kl-
tuspin
as
mol
1021.
(2-6)x10'
rate
M-I s -l
constant
rate
c221
[15].
can be strongly
and even
kinetic
in PDT, it
the
photosensitizers.
basic
competitive
determining
activation
feature
tendency they
at
are
at higher
accumulation
of
under
cer-
MT0 mechanism to be studied
the
has an important
as a search
So far,
increased
C18-
transplanted
(0.5-1.5)~10-~
the actual rate of the 3 2 the HP + Rad' interaction
PDT can be formulated
(iii)
sources
interesting
mechanisms
(i)
tissues
future.
various
tions:
in
might
in our Laboratemperature)
magnitude
controlled
of
in
normal
However,
depends on the Beyond
stages of
MT0 mechanism
point
conditions.
in detail
literature
the
in
recently nitrogen
range
order
tissues
k m- MT0 kl
a diffusion
Therefore,
cancer
concentra-
cases:
(WTT)max
to
the
COMMUNICATIONS
radical
values
early
in
'MT0
According
in
performed (at liquid
the
varies
RESEARCH
peroxy
corresponding
C2.11 that
mice
BIOPHYSICAL
and/or
Kinetic ESR spectroscopy in frozen tissue samples
mors
of
AND
practical
present for
in the
novel
tailored
wavelengths;
of
the
consedevelopment
and more efficient
to
in malignant
role
following (ii)
indica-
low toxicity;
tissues;
(iv)
optimal
photobleaching. If sidered tizers
the
MT0 mechanism will
likely
to
play
to be used in
is expected cluding
to
react
a probable
extending
the
be strongly
a role,
a new requirement PDT can be introduced: its as an efficient
decomposition
role
of
supported
radical during
and con-
for photosensiactivated form
"scavenger"
photoactivation
inand thus
Hp as a carrier.
REFERENCES 1.
D. Russell,
2.
Plenum, 1983, Y. Hayata, T. vative
T.
Dougherty: p. 160) Dougherty:
in Cancer
(Tokyo,
Adv.
Exp.
Med.
Biol.
(New York
Lasers and Haematoporphyrin Igaku-Shoin, 1984) 1035
Deri-
Vol.
186,
3.
4.
No.
2, 1992
G. Jori:
In
(New
York
Land,
T.
H.
6.
Abstracts.
van
dynamic
C.
Press.
T.G.
BIOPHYSICAL
Photoprocesses Eds:
G. Truscott,
den
RESEARCH
in
R.V.
1985,
pp.
Truscott:
Bergh: 3rd
COMMUNICATIONS
Biology
and Medicine
Bensasson,
G. Jori,
E.J.
349-355)
Inst.
Biennal
J.D.
J.
p.
183,
in
Radiat.
Britain,
Meeting
of
(Buffalo,
Spikes:
Emiliani,
490
Chemistry
Association
G. Jori, Smith,
9.
Plenum
AND
Biol.,
2,
421-
(1986)
5.
8.
Primary
R. Potter, 452
7.
BIOCHEMICAL
New York
the
pp.
430-439.
International
New York
Topics in Plenum,
M. Delmelle:
1986,
Photo-
1990)
Photodynamics 1984)
Photochem.
(Ed.:
Photobiol.,
K.C.
37,
487-
(1983)
G.R. cine
Buettner: Primary Photoprocesses (Eds: R.V. Bensasson, G. Jori,
Truscott, 10.
J.
Moan:
11.
R.
Bonnett,
New York
Plenum
Photochem.
pp.
Photobiol.,
C. Lambert,
Sinclair,
Press,
T.G.
Truscott:
D.G.
Whitten:
in E.J.
341-344,
2,
E.J.
Biology and MediLand and T.G. 1985)
445-449
Land,
P.A.
Photochem.
(1984)
Scourides,
R.S.
Photobiol.,
2,
l-8
(1983) 12.
M. Krieg,
13.
T.
Vidbczy,
14.
A.
Seret,
145-155 15.
A.
16.
H.
S.
T.
19.
21.
Obi:
J.
Durai,
P.K.
Das,
S. Raja
Chem.
(1967);
de Vorst:
22.
(1984)
publication
Kinichi
Phys.
V.S.
Lett.,
Mosyenko,
137, A.D.
J.
Photochem.,
Phys.
Chem.,
Mulay, (1967);
J.
Duchesne,
Phys.
Med.
L.N. N-1.
danov, N.N. Zoz: T. Shulykovskaya, R.W. Redmond, 523-529 (1988)
S.E.
G.L.
255-260 Bullach:
of Oxygen in Biological 1974, pp. 191-200, in
Kinetics of N.M. Emanuel: (Naukai Moscow, 1977, pp. C.M. Dettmer, D.H. Driscoll,
I.L. 743
235-252
38, 95,
(1991)
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