Skin Plasma Kidneys Liver B B B ox PB-Alb O2O2 1O21O2 + PB B ox PB Process 2 Process 1 O2O2 Urine Bile- intestine B = bilirubinAlb = albumin PB.

Copyright

All images in this presentation are the property of Jane Hanrahan unless otherwise referenced.

References http://omlc.ogi.edu/spectra/PhotochemCAD/html/bilirubin.html, accessed 14/07/06 McDonagh et al., Science, 208, 1980, 145-151. http://www.psoriasis.org/about/psoriasis/ Accessed 14/070/06 Edelson, R. L. Scientific American, 68-75, 1988. http://www.bundp.net/iframes/qlight/qlight_text_penetration.htm Accessed 15/07/06 http://www.fda.gov/ohrms/dockets/ac/03/briefing/3983B2_02_PhotoCure-Briefing%2

0Document.htm Accessed 15/0706

http://www.photocure.com/professionals/display.asp?xmlID=169 Accessed 15/07/06 http://www.photocure.com/professionals/ accessed 18/6/2004 http://www.novartispharma.at/download/info_material/visudyne_06.pdf Accessed

15/07/06. http://www.novartispharma.at/download/info_material/visudyne_06.pdf Accessed

15/07/06.

Photochemotherapy

A/Prof Jane Hanrahan

janeh@pharm.usyd.edu.au

Therapy with Radiation

Low energy (non-ionising) radiation - UV and visible light Phototherapy - natural chromophores as light

absorber Endogenous chemical absorbs light eg bilirubin

Photochemotherapy - added photosensitiser Exogenous chemical also required, eg PUVA, PDT

High energy (ionising radiation) - and X-rays Radiotherapy

Ionising radiation acts directly to kill cancer Radiotherapy with radiation sensitiser

Radiochemotherapy - Exogenous chemical also required

Phototherapy Treatment for Neonatal Jaundice

Immature liver unable to metabolise bilirubin

Glucuronyl transferase activity in new born is low

Accumulation of potentially toxic yellow lipophilic bilirubin accumulates in serum leads to Jaundice

0.1 -1.5mg/100ml is normal 15-25mg/100ml indicates hyperbilirubinemia

Elimination of Bilirubin

Skin Plasma

Kidneys

Liver

B

BBox

PB-Alb

O2

1O2

+

PB

Box

PB

Process 2

Process 1

O2

Urine

Bile-intestine

B = bilirubin Alb = albuminPB = mixture of photoisomers of bilirubinBox = mixture of photooxidaton products of bilirubin

h

Absorption spectrum of

Bilirubin

Wavelength (nm)

60,000

40,000

20,000

200 300 400 500 600 700

Mola

r exti

nct

ion c

oeffi

cient

http://omlc.ogi.edu/spectra/PhotochemCAD/html/bilirubin.html, accessed 14/07/06

E-Z isomerism of Bilirubin

McDonagh et al., Science, 208, 1980, 145-151.

PUVA Therapy

Psoralen-UVA therapyUsed in the treatment of psoriasis, vitiligo

and cutaneous T-cell lymphoma (CTCL)All diseases are characterised by rapid

proliferation of cellsFormation of adducts in DNA results in

inhibition of DNA synthesis resulting in cell death or slower cell growth

Can increase risk of basal cell carcinoma

Psoriasis

http://www.psoriasis.org/about/psoriasis/ Accessed 14/070/06

Psoralens

Psoralens are a furocoumarin derivatives

Found in nature in figs, limes, parsnip roots and other fruit and vegetables

Absorb light between 250-400 nm because of extended conjugation

UVA absorbance due to the pyrone moiety

Psoralens

8-Methoxypsoralen (8-MOP)5-Methoxypsoralen (5-MOP)

4,5’,8-Trimethylpsoralen (TMP)

Oral PUVA

After oral administration of 8-MOP, patients become gradually reactive to UVA and therefore to photochemotherapeutic treatment. Patients are maximally reactive 2-3 hours after ingestion of the drug.

Treatment TimeU

VA

Sen

siti

vit

y

Hours1 2 3 4 5 6 7 8

25

0

50

75

100

MA

XIM

UM

SEN

SIT

IVIT

Y

Mechanism of Action

4’,5’

Edelson, R. L. Scientific American, 68-75, 1988.

Photoreaction between

Psoralens

and DNA Ground state intercalation of psoralen in the dark Absorption of a photon by the 4’,5’-double bond or

3,4-double bond of psoralen Photoreaction between 4’,5’-double bond or the

3,4-double bond of the furan ring of the psoralen molecule and the 5,6-double bond of the pyrimidine base (thymine) to give a monofunctional adduct

Absorption of a photon by the 3,4-double bond of psoralen in the 4’,5’- monofunctional adduct

Photoreaction between 3,4-double bond of the pyrone ring of the psoralen molecule and the 5,6-double bond of a second pyrimidne base (thymine) giving a bifunctional adduct

Interaction between 8-MOP

and DNAThymine 8-Methoxypsoralen

Dark Weak Intercalation

UV-A

UV-A

4’,5’-Monoadduct 3,4-Monoadductor

Effects of PUVA

Phototoxic effect on skin

ErythemaInduction of dark pigmentation

Antiproliferative effect

Inhibition of growth or lethal effect (at high doses) on bacterial and mammalian cellsInhibition of DNA, RNA and protein synthesisInhibition of synthesis of epidermal DNA in mouse skinInhibition of infectivity of DNA viruses

Mutagenic effect

Causes mutations in a variety of micro-organisms & cell lines eg Eschericia coli, Sacchromyces cerevisiae, Sarcina lutea, Salmonella typhimurium & Chinese hamster cells

Photocarcinogenic activity

Photocarcinogenic activity on mouse skinRecent studies show increase in BCC in PUVA patientsInhibition of EGF cell binding

Other effects Alteration of immune system

Photodegradation of 8-MOP

Photodynamic Therapy (PDT)

Evolving therapy for treatment of cancers, psoriasis, vascular occlusion and macular degeneration

Involves administration of a photosensitiser followed by irradiation with visible light

Photosensitiser is often a porphyrin derivative Optimal tissue penetration occurs between 650-

800 nmUptake of photosensitisers is selective for

tumour cellsFirst generation photosensitisers have

several problems in practiceMany second generation photosensitisers

currently in clinical trials

Light penetration of skin

http://www.bundp.net/iframes/qlight/qlight_text_penetration.htm Accessed 15/07/06

PDT Mechanism

Edelson, R. L. Scientific American, 68-75, 1988.

Haematoporphyrin

• HpD is a mixture of Hp derivatives where

R1= R2=

5-Aminolevulinic acid (ALA)Stimulates the synthesis protoporphyrin IX,

(PpIX) the immediate precursor to haem and an endogenous photosensitiser

Can be administered systemically or topically

Optimal concentration reached in 2-4 h max = 630nm Concentration in tumour can be measured

using fluorescenceRapidly undergoes photobleachingHydrophillic nature of ALA restricts skin

penetration -this can be overcome by use of esters

eg Metvix, Methyl ALA, approved for non-melanoma skin cancers and pre-cancerous growth in many countries

Synthesis of Protoporphyrin

IX

PpIX rate of formation is dependent on synthesis of ALA from glycine and succinyl CoA which is governed by -ve feedback by the concentration of free haem. Since conversion of PpIX to haem is relatively slow, administration of exogenous ALA bypasses the -ve feedback.

Haem synthesis in tumour cells is slower, allows PpIX to accumulate

http://www.photocure.com/professionals/ accessed 18/6/2004

http://www.photocure.com/professionals/ accessed 18/6/2004

http://www.photocure.com/professionals/ accessed 18/6/2004

Verteporfin (Visudyne)

Used for the treatment of wet age-related macular degeneration

A chlorin-type porphyrinUsed with laser at 689 nm

http://www.novartispharma.at/download/info_material/visudyne_06.pdf Accessed 15/07/06.

Absorbance of Verteporfin

http://www.novartispharma.at/download/info_material/visudyne_06.pdf Accessed 15/07/06.

Potential Uses of PDT

• Actinic keratosis

• Bowen’s disease

• Superficial Basal cell carcinoma

• Superficial Squamous cell carcinoma

• Keratocanthoma

• Bladder cancer

• AMD

• Psoriasis, acne• Hair removal• Warts• Non-small cell lung

cancer• Metastatic breast

cancer• Brain tumours• Post angioplasty

restenosis

Radiotherapy

Two critical cell targets for radiation

DNA, to directly cause double strand breakage Water, causing the formation of free radicals in the

cell’s aqueous environment, leading to radical attack on DNA

Radiotherapy

Different radiation types have different energy deposition rates or Linear Energy Transfer (LET) which lead to different Relative Biological Efficiencies (RBE)

a + b radiation have high LET g + X rays have low LET

Clonogenic cell survival curves for cultured mammalian tumour cells irradiated with increasing doses of different quality LET beams. The RBE decreases with dose. The surviving fraction is measured by soft agar assay

100

10

1

1 2 3 4 5 6 7 8

RBE 5.6

RBE 3.3

Low LETHigh LET

Radiation dose (Gy)

Surviving fraction%

Irradiated water

Physical Stage

H2O H2O+ + H2O* + e-

H2O+ + H2O H2O+ + •OH

H2O+ + e-

H2O* H• + •OH

H2 + O•

radiation

Irradiated Water

Chemical Stage

•OH + •OH H2O2

•OH + e- -OH

•OH + H• H2O

H3O+ + e-aq H• + H2O

H• + H• H2

Radiation Therapy & Oxygen

Oxygen is an important mediator because it reacts efficiently with free radicals to “fix” the damage

Oxygen enhancement ration (OER) compares cell kill with and without Oxygen

Radiation sensitiser (oxygen mimetic) needed for hypoxic tumours

Radiation sensitisers

Misonidazole Razoxane

Summary

Treatment of neonatal jaundice PUVA therapy for psoriasis Photodynamic therapy Radiochemotherapy of tumours