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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.
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
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