porfirinas e aplicaÇÕes biolÓgicas departamento de química, universidade de aveiro josé a. s....
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PORFIRINASPORFIRINAS
E APLICAÇÕES BIOLÓGICASE APLICAÇÕES BIOLÓGICAS
Departamento de Química, Universidade de Aveiro
José A. S. José A. S.
CavaleiroCavaleiro
Complex mixture of haematoporphyrin oligomers (dimers – hexamers)
PhotofrinPhotofrin
HN
N
N
NH
HO2C CO2H
OH
OH
Photosensitisers’ medicinal Photosensitisers’ medicinal applicationsapplications
1 25
3
1O2
3O2
6
1 – Light absorption2 - Fluorescence3 – Internal conversion 4 – Intersystem Crossing, ISC5 - Phosphorescence6 - Type II Mechanism7 - Type I Mechanism
7 Radical orions radicals
En
erg
y
oS
S1
S2
Sn
4
T1
Modified Jablonski Diagram
1867, Thudichum
1910, Hausmann
1913, Meyer-Betz
1924, Policard
1942, Auler and Banzer
Fluorescence properties of haematoporphyrin (Hp)
Effect of Hp and light on red blood cells; skin reactions in mice exposed to light after Hp administration (photosensitivity, phototoxicity)
1st report of human photosensitization by porphyrins
(he injected himself with 200 mg of Hp; subsequently noticed prolonged pain and swelling in light-exposed areas)
1st report of fluorescent porphyrin localization in a malignant tumour
(he observed the Hp red fluorescence in a rat sarcoma illuminated with UV light)
Localization and fluorescence of exogenously administrated porphyrins in malignant tumours
1948, Figge and Weiland
1951, Manganiello and Figge
1955, Figge et al.
Tumour localizing properties of porphyrins and application in the diagnosis and treatment of tumours.
240 mice with induced and transplanted tumours
50 non-tumours bearing mice Porphyrin localization in each tumour-type; fluorescence within 24-28h of administration; 10-14 days persistence.
Hp effects in 3 patients with head and neck malignancies
(no fluorescence detected; lower doses in humans – 30-120 mg)
Intravenous administration of Hp dihydrochloride to patients before excision of variety of benign and malignant lesions.
Fluorescence was observed and increased in proportion to Hp dose. Higher doses allowed a breast cancer detection.
1955, Peck Hp fluorescence in biliary and bladder systems (in animals and humans); with a high dosage (1000 mg) one patient exhibited fluorescence in a cervical carcinoma
Hp can play a role as a diagnostic tool for cancer
But... a major disadvantage was the large dose of photosensitiser, which led to phototoxicity.
1955, Schwartz
1960, Lipson
Used Hp was a mixture of porphyrins; after purification, “pure” Hp localized poorly in tumours, but residue left behind showed great affinity for tumours tissues.
Hp derivative (HpD) described.
Hp-dihydrochloride + sulphuric /acetic acid, precipitation with sodium acetate. The precipitated + NaOH and pH adjusted to 7.4.
HN
N
N
NH
CO2HHO2C
OH
OH
HpD, Stage II
5% H2SO4-AcOH
Haematoporphyrin
15 min, rtHpD, Stage I
Alkali treatment
(then adjusted to pH 7.4)
1976, Kelly The 1st published account of clinical PDT with HpDhuman bladder tumour cells transplanted into mice could be destroyed using PDT
1978, Dougherty The 1st extensive clinical survey with HpD
25 patients with cutaneous or sub-cutaneous malignancies
1993 Canadian authorities granted the 1st regulatory approval (bladder, oesophagus) for cancer PDT using Photofrin
(commercialised by QLT Phototherapeutics, Vancouver)
France (oesophagus, bladder);
Germany (lung);
Japan (lung, oesophagus, stomach,
cervix);
Netherlands (lung, oesophagus);
USA (oesophagus, lung)
Subsequent approvals:
In Photodynamics News, Vol. 4 Number 3, 2001In Photodynamics News, Vol. 4 Number 3, 2001
In Photodynamics News, Vol. 4 Number 3, 2001In Photodynamics News, Vol. 4 Number 3, 2001
PDT StudiesPDT StudiesHN
N
N
NH
NH
O
HN
N
N
NH
OCH3
OCH3
H3CO
HN
N
N
NH
NH
O
HN
N
N
NH
OCH3
OCH3
H3CO
Survival of S.aureus ATCC cells incubated for 10 min. with different concentrations of porphyrins and irradiated for 30 min with white light (50 mW/cm2)
0 1 10
Porphyrin (µM)
10-5
10-4
10-3
10-2
10-1
100
101
102
16 16 + light 24 24 + light 25 25 + light
% S
urvi
val
Inactivation results:Inactivation results:
HNNH
HNNH
OO
NH
O
N
N
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
CH3CH3
CH3CH3
CH3
CH3
HNNH
HNNH
OO
NH
O
NH2
NH2
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
HN
N
N
NH
N
N
N
O
OH
CH3
H3C
CH3
n I -
3 I -
3 I -
HNNH
HNNH
OO
NH
O
N
N
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
CH3CH3
CH3CH3
CH3
CH3
HNNH
HNNH
OO
NH
O
NH2
NH2
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
HN
N
N
NH
N
N
N
O
OH
CH3
H3C
CH3
n I -
3 I -
3 I -
0 1 10
10-5
10-4
10-3
10-2
10-1
100
101
102
16 16 + light 24 24 + light 25 25 + light
Surv
ival (
%)
Porphyrin (µM)
Survival of E. coli cells incubated for 10 min with different concentrationsof porphyrins and irradiated for 30 min with white light (50 mW/cm2)
HNNH
HNNH
OO
NH
O
N
N
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
CH3CH3
CH3CH3
CH3
CH3
HNNH
HNNH
OO
NH
O
NH2
NH2
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
HN
N
N
NH
N
N
N
O
OH
CH3
H3C
CH3
n I -
3 I -
3 I -
HNNH
HNNH
OO
NH
O
N
N
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
CH3CH3
CH3CH3
CH3
CH3
HNNH
HNNH
OO
NH
O
NH2
NH2
HN
N
N
NH
N
N
N
O
CH3
H3C
CH3
HN
N
N
NH
N
N
N
O
OH
CH3
H3C
CH3
n I -
3 I -
3 I -
Inactivation results:Inactivation results:
PORFIRINASPORFIRINASCOMO AGENTESCOMO AGENTES
ANTIVIRAIS E ANTIFÚNGICOSANTIVIRAIS E ANTIFÚNGICOS
Síntese e aplicação de porfirinas em formulações Síntese e aplicação de porfirinas em formulações antivirais (pat. Nº 102 572)antivirais (pat. Nº 102 572)
Aplicação de porfirinas em formulações Aplicação de porfirinas em formulações antifúngicas (pat. Nº 102 581)antifúngicas (pat. Nº 102 581)
http://herpes.on.net/genital_herpes/details.htmhttp://herpes.on.net/genital_herpes/details.htm
NH
N
N
NHO
F
F
F F
F
F
F F
F
O
O
O
O
CH2
O
O
F
F F
F
HO
HO
CH2
OH
O
O
OH
R
H
HOO
HOHO
OH
AcOO
AcOOAc
OAc
20
2
3
4
R
1
HN
N
N
NHFF
F
F F F F
FF
F
FF
F
F
F
FF
F
F
O
O
OO
O
O
OH
HOOH
O
O
R
F
OH
7
5
6
R HN
N
N
NH O
O
O
O
O
CH2
O
O
OH
HO
CH2
OH
O
R
H
OH
10
9
8
R
HN
N
N
NH
N
N
N
O
O
O
O
O
CH2
O
O
OH
HO
CH2
OH
O
R
H
OH
11
15
14
R
O
O
O
CH2
O
O
OH
HO
CH2
OH
O
H
HN
N
N
NH
N
N
N
H3C
CH3
CH3
O
O R
OH
16
18
17
R
+
. 3 I -
+
+
PORFIRINAS COMO AGENTES ANTIVIRAISPORFIRINAS COMO AGENTES ANTIVIRAIS
Effect on Plaque FormationEffect on Plaque Formation
0
20
40
60
80
100
Inhibition
%
1 2 3 4 20 21 5 6 7 8 9 10 11 14 15 16 17 18
porphyrins
HSV-1
HSV-2
0
20
40
60
80
100
Inhibition
%
1 2 3 4 20 21 5 6 7 8 9 10 11 14 15 16 17 18
porphyrins
HSV-1
HSV-2
Virucidal EffectVirucidal Effect
N CH3
N
N
N
N
R
R
R
R M
N
N
C5H11
N CH3
2H1
2H3
R
Zn
Porf.
4
M
+I-
+ I-
2 2H+ I-
PORFIRINAS COMO AGENTES ANTIFÚNGICOSPORFIRINAS COMO AGENTES ANTIFÚNGICOS
0
10
20
30
40
50
60
70
CM
I (m
g/m
L)
2 3 4
porfirinas
C. albicansC. glabrata
C. kruseiC. tropicalis
C. parapsilosisC. guilliermondii
Cr. neoformansT. cutaneum
C. krusei (6258 ATCC)C. parapsilosis (90018 ATCC)
Actividade sobre fungos leveduriformes Actividade sobre fungos leveduriformes
Actividade sobre fungos filamentosos Actividade sobre fungos filamentosos
0
10
20
30
40
50
60
70
CM
I (m
g/m
L)
Porf. 3 Porf. 4
Porfirinas
A. niger
A. f lavus
A. fumigatus
S. brevicaulis
F. oxysporum
T. soudanense
A. fumigatus (7097 ATCC)
A. fumigatus (7100 ATCC)