PORFIRINASPORFIRINASE 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

Ener

gy

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, 2001

In Photodynamics News, Vol. 4 Number 3, 2001

PDT StudiesPDT StudiesHN

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 -

0 1 10

10-510-4

10-3

10-2

10-1

100

101

102

16 16 + light 24 24 + light 25 25 + light

Sur

viva

l (%

)

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 -

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 18porphyrins

HSV-1HSV-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-1HSV-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. glabrataC. kruseiC. tropicalisC. parapsilosisC. guilliermondiiCr. neoformansT. cutaneumC. 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)