Copyright

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

References1. http://medlib.med.utah.edu/kw/derm/pages/meet_2.htm

accessed 16/7/062. D. E. Moore, Drug Safety, 25, 345-372 (2002)

After completion of these lectures you should be able

to:

1. Describe the effects that the different classes of UV radiation have on the skin at the earths surface.

2. Be able to describe structural features common to drugs that result in photosensitivity reactions.

3. Explain the two different mechanisms of photodynamic action and the role in which biological molecules paly in photodynamic action.

4. Understand the difference between photoallergy and phototoxicity.5. Describe how certain NSAIDS cause photosensitivity reactions and understand how this is

related to their structure.6. Explain the cellular defence mechanisms that biological systems use to protect themselves

from UV radiation.7. Describe the ideal properties of sunscreens and explain the mechanisms by which various

sunscreens are effective.8. Understand how phototherapy is effective in treating neo-natal jaundice.9. Be able to describe the mechanism through which psoralens in conjunction with UVA

radiation are effective in treating skin diseases such as psoriasis.10. Describe the ideal properties of PDT drugs and their use in the treatment of various cancers

and skin diseases. Understand the mechanism by which these drugs act.11. Be able to explain the role of oxygen and radiation sensitisers in the radiotherapy of

tumours.

Radiation Effects

A/Prof Jane Hanrahanjaneh@pharm.usyd.edu.au

Objectives

Describe the effects of low-energy (non-ionising) radiation on the human body.Sunburn

Understand how sunscreens work

Drug induced photosensitivity - phototoxicity and photoallergy

Suntan Exposure restricted to minimal erythemal dose

(MED) Thickening of epidermis Darkening of existing pigmentation New pigment formation

Melanin Proliferative changes in epidermis Suppression of T-lymphocytes

Sunburn

Stage 1 Immediate faint erythema

Occurs during exposure - disappears quickly

Stage 2 Delayed erythemal exposure

(2-4 h, peaks at 14-20 h, persists 24-48 h)

Sunburn

Stage 3 Dead cells form a desquamating layer

Desquamation begins 72-96 h

Stage 4 Increase in DOPA +vemelanocytes

Stage 5 Repeated sunburn skin cancer

Sunburn

Markers Melanin tanning Langerhans cells

(macrophage - like cells of the epidermis) Ornithinedecarboxylase activity

Action Spectrum for induction of

erythema

Action spectrum (approximate) for the induction of erythema in human skin, and average intensity at the earth’s surface. The figure also depicts the ranges of various UV regions. D. E. Moore, Drug Safety, 25, 345-372 (2002)

Skin Layers and UV Light Penetration

(200-280nm) blocked by ozone layerProduced by some artificial light sourcesMay penetrate top layers of skin(280-320nm) responsible for sunburnContributes little to tanningCauses squamous cell carcinoma and Leathery skin(320-400nm) penetrates deep into theDermis.Causes wrinkles, blotches & age-spotsStimulate melanin to tanContributes to sunburn

200 250 300

UVC

UVB

UVA

http://medlib.med.utah.edu/kw/derm/pages/meet_2.htm accessed 16/7/06

Penetration and effects of

UV radiation

Absorption spectrum of DNA &

protein

Absorption spectra of DNA (calf thymus) and protein (bovine serum albumin at equal concentrations (20 µg/ml). D. E. Moore, Drug Safety, 25, 345-372 (2002)

UV chromophores in the skin

D. E. Moore, Drug Safety, 25, 345-372 (2002)

Clinical Phototoxic Responses

Strong delayed erythema Onset 8-24 hours lasts 2-4 days May be darker than normal sunburn egpsoralens

More rapid, transient erythema Immediate onset, lasts 1-2 days No oedema, localised burning and itching Eg coal tar derivatives, anthracene, acridine

Rapid transient wheals and flares Eghaematoporphyrin (cancer treatment)

ADRAC (Australia)Reports to December 1994

Diuretic Agents Moduretic (Hydrochlorothiazide + Amiloride) 320 Dyazide (Hydrochlorothiazide + Triamterene) 156 Furosemide (furosemide) 148 Hydrochlorothiazide 72 Chlorothiazide 27

Other Combination Formulations Bactrim/Septrin 204 (sulfamethoxazole + trimethoprim) Tetracycline + Nystatin 66

Attempted differentiation

between phototoxicity and

photoallergyObservation Result in

PhototoxicityResult in Photoallergy

Reaction to first exposure Present Absent

Latency between exposure & response Variable May occur

Gross reactions to structurally related compounds

Absent Varied

Clinical changes Like sunburn Varied

Flares at previously involved sites Never Possible

Development of persistent light reaction

Never Rarely

Incidence for a given compound Very high Usually low

Concentration of drug required for reaction

High Low

Action spectrum & absorption spectrum

Normally similar

Action spectrum at longer

Results of photo patch Immediate Delayed

Examples of photosensitising drugs

Sulfonamides Furocoumarins

Chlorodiazepoxide

Methyldopa

Protriptylline

Examples of photosensitising drugs

Norethisterone Nalidixic acid

Tetracyclines

Chlorpromazine Thiazides

Common structural features of

Photosensitisers

Low molecular weight (200-500)

Planar, Tricyclic or Polycyclic Often contain heteroatoms

Stabilise resonance structures All absorb UV/visible radiation

Photosensitised Reaction Mechanism

Do 1D

3D

1O2 + Do

Do + A• H•

D- + A• H+

orA-OO-H

AH3O2

absorption

fluorescence orInternal conversion

singlet excited state

triplet excited state

singlet oxygen

intersystem crossings

phosphorescenceor intersystem crossings

ground stateabsorbs light

AH

e-promoted to next energy levele- spin state not changed

O2 in ground state, exists as a triplet

peroxy molecule

Type IIType I

Free radical

Photodynamic action

Type 1 Energy is sufficient to cause dissociation of

reaction molecule loses H

Free radical species - reactive often undergoes reaction with 3O2 to produce a

peroxidised molecule chain reaction

Type 2 Photosensitised oxidation

Photodynamic Action

Internal conversion Interaction between energy levels Molecules can dissipate energy via vibration

relaxation, returns to original state No chemical change to molecule

Fluorescence Molecule gives up photon of light as it returns

to ground statePhosphoresence

Molecule gives up photon of light as it returns to ground state from triplet excited state

Acceptors for Photodynamic

ActionProteins - amino acids eghistidine

Tryptophan - types I (free radical) and types II 1O2

Type II reaction

Imidazole ring reacts with 1O2 adding O2 across the double bond, forming an unstable cyclic intermediate which subsequently breaks down

Histidine in protein gets damaged

+

Acceptors for Photodynamic

ActionLipids

Type II reaction1O2adds across double bond of lipid molecule Lipids are found in cell membrane, reaction leads to disruption of the cell membrane and cell death

Acceptors for Photodynamic

ActionCarbohydrates - alcohols, sugars, vitamin C

Type I reactionContain many hydroxyls (-OH) which interact with the triplet excited state giving a free radical reactionA free radical is formed and can react further

alcohol ketone

Acceptors for Photodynamic

ActionNucleic Acids - Purines

Guanine, xanthine + 1O2

Purines can be oxidised and therefore are susceptible to attack by singlet oxygen.Not as susceptible as histidine or tryptophan, but damage does occur

Type II damage

Photosensitisers Absorbs Sunlight Energy Pathological response

in Skin

Endogenous Photosensitisers» Porphyrins complex with Fe in Haemoglobin» Disease state - Porphyria (excess unbound porphyrins

in circulation (400-450 nm)

Exogenous Photosensitisers» Contact

Tars and psoralens, perfumes, plants» Systemic

Psoralens and drugs (320-400 nm)

Photochemical Activity and

Phototoxicity

Drug or pollutant

1O2prod. (1)

Free radical generation (2)

Phototoxicity in Mouse (3)

Clinical Reports of Phototoxicity (4)

8-Methoxypsoralen

37 39 strong Very many

Cloropromazine 54 36 strong many

Pomazine 20 17 moderate few

Hydrochlorothiazide

13 14 strong many

Frusemide 40 22 strong many

Nalidixic acid 200 18 strong many

Cloroquine 18 14 weak few

Diazepam 4 <0.1 weak few

Chlorodiazepoxide

2 <0.1 weak few

Qinine 96 2 weak few

Metronidazole 0 scavenger

moderate few

Azathioprine 1 scavenger

moderate many

Benoxaprofen 37 42 strong Very many

Naproxen 15 11 moderate some

Indometacin <1 <0.1 none few

Oxytetracycline 11 0.5 moderate some

Demeclocycline 13 5 strong many

Dimethylbenzantracene

232 3 Strong (topical)

many

Benzacridine 185 2 strong manyComparison of fundamental photochemical activity with mouse

phototoxicity tests and clinical photosensitivity responses(1) Based on O2 uptake and flash photolysis measurements. (2) Based on polymerisation and electron spin resonance experiments. (3) Adapted from various literature reports (4) Adapted from ADRAC and Magnus

Photsensitisation

UV + Photosensitising Chemical

Excited State Chemical

Loss of Energy by Fluorescence Phosphorescence Internal Conversion

Photochemical Reactions

Photochemical Reactions

1. Energy transfer to molecular oxygenExcited singlet oxygen oxidation of lipids or proteins

2. Energy transfer to BiomoleculeOxidation of excited state biomolecules

3. Covalent binding to BiomoleculeAltered DNA, Lipid or Protein

4. Formation of Toxic Photoproduct

5. Binding to skin proteinFormation of Hapten as photoproductImmunological Sensitisation

Photsensitising Drug

Excited State Drug

Triplet State

Energy transfer to molecular O2

Excited singlet O2

Oxidation-peroxidation of biomolecule (lipid, protein)

Energy transfer to biomolecule

Oxidation of excited state biomolecule

Molecular change

Free Radicals

Formation of photoproduct(s)

Electron transfer or covalent binding to biomolecule

Molecular change to cell components

Photo-oxidation of cell components

Toxic reaction with cell components

Damage to critical cell components

Phototoxic Skin Response

Deactivation Mechanisms(Fluorescence, Internal conversion, etc)h

h

Naproxen

Peroxy radical

radical

Benoxaprofen

radical

Hydroxyl radical

Diclophenac

carbazole

photosubstitution

photoreduction

Secondary photoproduct

NSAIDS

Naproxen Ketoprofen Ibuprofen

SuprofenBenoxprofen

SulindacIndomethacin

Diflusinal

Diclofenac Piroxicam

Cellular Defence Mechanisms

Vitamin C and E Natural antioxidants

Uric AcidGlutathione

Protects mitochondria from free radicalsSuperoxide Dismutase

Catalyses reaction of toxic superoxide radical O2

-. to O2

Catalase Reduces hydrogen peroxide (HOOH)

Various oxidase - reductase systems

Cellular Defences

MechanismsVitamin A, carotenes

Contain long chains of conjugated double bonds

Other dietary Supplements Flavonoids

Depletion of defence mechanisms may lead to damage to cell macromolecules eg DNA

Can drugs initiate skin

cancer?

Repeated phototoxic injury leads to skin cancer in experimental animals

PUVA treatment (psoralens + UVA radiation) for psoriasis can cause skin cancer after prolonged use

increase in BCC

Immunosuppressive therapy with azathioprine is associated with high cancer incidence

In general population, phototoxic reactions may add to the already high risk of skin cancer from sunburn

Summary

Action of UV on biochemicals, eg proteins, DNA, sugars

Interaction of drugs with UV radiation resulting in photosensitisation or photoallergy

Photodynamic action Type 1 Type 2

Cellular defence mechanisms