1. Angela Dann Monday, October 9, 2006
Photodynamic Therapy of Cancer: The Design and Characterization of Photosensitizing Agents
Angela Dann
Monday, October 9, 2006

2. Photosensitizing Agents
History
Introduction
Process of Photodynamic therapy (PDT)
PDT to treat cancer
Photosensitizing Agents
Requirements
Advancements
Trials using PDT on tumor cells
Conclusions
Future applications

3. History Light used as therapeutic agent for 3000+ years
Egyptian, Indian, and Chinese civilizations
Psoriasis, rickets, vitiligo, skin cancer
Photodynamic Therapy (PDT) developed within the last century
Nature 2003, 3, 380.

4. History
Nature 2003, 3, 380.

5. History Niels Finsen (late 19th century) Oscar Rabb (100+ years ago)
Red light to prevent formation and discharge of small pox postules
UV light from the sun to treat cutaneous tuberculosis
Nobel Prize 1903
Oscar Rabb (100+ years ago)
Acridine in combination with certain wavelengths of light
Lethal to infusoria
Nature 2003, 3, 380.

6. History Herman Von Tappeiner, A. Jesionek W. Hausmann
Defined photodynamic action
Topically applied eosin and white light
W. Hausmann
1st studies with haematoporphyrin and light
Killed paramecium and red blood cells
Friedrich Meyer-Betz (1913)
1st to treat humans with porphyrins
Haematoporphyrin applied to skin, causing swelling/pain with light exposure
Nature 2003, 3, 380.

7. History Samuel Schwartz (1960’s) Lipson, E.J. Baldes I. Diamond (1972)
Developed haematoporphyrin derivative (HpD)
Haematoporphyrin treated with acetic and sulfuric acids, neutralized with sodium acetate
Lipson, E.J. Baldes
HpD localization in tumor cells, fluorescence
I. Diamond (1972)
Use PDT to treat cancer
Nature 2003, 3, 380.

8. History Thomas Dougherty (1975) J.F. Kelly (1976) Canada (1999)
HpD and red light
Eradicated mammary tumor growth in mice
J.F. Kelly (1976)
1st human trials using HpD
Bladder cancer
Canada (1999)
1st PDT drug approved
Nature 2003, 3, 380.

9. Introduction: Process of Photodynamic therapy
Two individually non-toxic components brought together to cause harmful effects on cells and tissues
Photosensitizing
agent
Light of specific
wavelength
Nature 2003, 3, 380.

10. Introduction: Reaction Mechanisms
Type 1:
Direct reaction with substrate (cell membrane or molecule)
Transfer of H atom to form radicals
Radicals react with O2 to form oxygenated products
Type 2:
Transfer of energy to O2 to form 1O2
Nature 2003, 3, 380.

11. Introduction: Reaction Mechanisms
Ratio of Type 1/Type 2 depends on:
Photosensitizing agent, concentration of substrate and O2, binding affinity of photosensitizing agent to substrate
Reactive oxygenated species (ROS)
Free radicals or 1O2
Half-life of 1O2 < 0.04 ms
Radius affected < 0.02 mm
Nature 2003, 3, 380.

12. Introduction: Type 1 and 2 Reactions
Nature 2003, 3, 380.

13. Introduction: Treatment of cancer
PDT best suited for:
Early stage tumors
Inoperable for various reasons
Limited success due to lack of specificity and potency of photosensitizing agents
Three mechanisms of tumor damage
Nature 2003, 3, 380.

14. Introduction: Mechanism 1
Direct Photodamage to Tumors by ROS
Problems:
Non-homogenous distribution of photosensitizing agent within tumor
Availability of O2 within tumor cells
Reduction of O2 presence during PDT
Overcoming O2 depletion:
Lower light fluence rate
Pulse light delivery – allow re-oxygenation
Nature 2003, 3, J. of Nuclear Medicine 2006, 47, 1119.

15. Introduction: Mechanism 2
Vascular Damage
Blood vessels supply nutrients to tumor cells
Effects:
Microvascular collapse
Tissue hypoxia and anoxia
Thrombus formation
Associated with halting tumor growth
Angiogenic factors upregulated
Nature 2003, 3, J. of Nuclear Medicine 2006, 47, 1119.

16. Introduction: Mechanism 3
Immune Response
Movement of lymphocytes, leukocytes, macrophages into treated tissue
Difference in reactions toward normal and tumor tissues
Upregulation of interleukin, not tumor necrosis factor-a
Neutrophil – slows tumor growth
Required to purge remaining cells
Nature 2003, 3, 380.

17. Photosensitizing Agents: Requirements
Selectivity to tumor cells
Photostability
Biological stability
Photochemical efficiency
No cytotoxicity in absence of light
Strong absorption – nm
Good tissue penetration
Long triplet excited state lifetime
J. of Photochemistry and Photobiology A: Chemistry 2002, 153, Photochemistry and Photobiology 2001, 74, 656.

18. Photosensitizing Agents: Classes
Porphyrin derivatives
Most widely used
Chlorins
Reduced porphyrins
Derivatives from chlorophyll or porphyrins
Phthalocyanines
2nd generation
Contain diamagnetic metal ion
Porphycenes
Synthetic porphyrins
Pharmaceutical Research 2000, 17, 1447.

19. Photosensitizing Agents: Examples
Photofrin
Foscan
5-Aminolevulinic acid (5-ALA)
Mono-L-aspartyl chlorin e6 (NPe6)
Phthalocyanines
Meso-tetra(hydroxyphenyl)porphyrins (mTHPP)
Texaphyrins
Tin ethyl etiopurpurin (SnET2, Purlytin)

20. Photosensitizing Agents: Photofrin
1st clinical approval (1999) in Canada
Bladder cancer treatment
Most commonly used photosensitizer
Destroys mitochondria
Dihematoporphyrin ether (DHE)
bis-1-[3(1-hydroxy-ethyl)deuteroporphyrin-8-yl] ethyl ether
Active component of HpD
Photochemistry and Photobiology 2001, 74, 656.

21. Photosensitizing Agents: Photofrin
Partially purified haematoporphyrin derivative (HpD)
Mixture of mono-, di-, and oligomers
Twice as phototoxic as crude haematoporphyrin (Hp)
Crude Hp consists of range of porphyrins
Convert to HpD by acetylation and reduction using acetic and sulfuric acids, filtering, and neutralizing with sodium acetate
Photochemistry and Photobiology 2001, 74, 656. Nature 2003, 3, 380.

22. Photosensitizing Agents: Photofrin
Limitations:
Contains 60 compounds
Difficult to reproduce composition
At 630 nm, molar absorption coefficient is low (1,170 M-1 cm-1)
Main absorption at 400 nm
High concentrations of drug and light needed
Not very selective toward tumor cells
Absorption by skin cells causes long-lasting photosensitivity (½ life = 452 hr)
Nature 2003, 3, J. of Photochemistry and Photobiology A: Chemistry 2002, 153, 245.

23. Photosensitizing Agents: Advancements
Need to overcome limitations of Photofrin
New photosensitizers developed according to ideal situations
Increase specificity to tumor cells
Increase potency
Decrease time of sensitivity to sunlight after treatment

24. Photosensitizing Agents: Foscan
Chlorin photosensitizing agent
Approved for treatment of head and neck cancer
Low drug dose (0.1 mg/kg body weight)
Low light dose (10 J/cm2)
Complications due to potency
Nature 2003, 3, 380.

25. Photosensitizing Agents: 5-Aminolevulinic acid (5-ALA)
Hydrophilic zwitterion at physiological pH
Approved for treatment of actinic keratosis and BCC of skin
Topical application most frequently used
Endogenous photosensitizing agent
5-ALA not directly photosensitizing
Creates porphyria-like syndrome
Precursor to protoporphyrin IX (PpIX)
Nature 2003, 3, 380. Photochemistry and Photobiology 2001, 74, 656. Pharmaceutical Res. 2000, 17, 1447.

26. Photosensitizing Agents: Mono-L-aspartyl chlorin e6 (NPe6)
2nd generation hydrophilic chlorin
Derived from chlorophyll a
Chemically pure
Absorption at 664 nm
Localizes in lysosomes (instead of mitochondria)
Reduced limitations compared to Photofrin
Decreased sensitivity to sunlight (1 week)
½ life = hr
Photodermatol Photoimmunol Photomed 2005, 21, 72.

27. Photosensitizing Agents: Phthalocyanines
2nd generation
Ring of 4 isoindole units linked by N-atoms
Stable chelates with metal cations
Sulfonate groups increase water solubility
Examples (AlPcS4, ZnPcS2)
Aluminum chlorophthalocyanine sulfonate
More prolonged photosensitization than HpD
Less skin sensitivity in sunlight
Photochemistry and Photobiology 2001, 74, 656. J. of Nuclear Medicine, 2006, 47, 1119.

28. Photosensitizing Agents: Phthalocyanines
Tetrasulfonated AlPcS4
Hydrophilic
Deposited in vascular stroma
Affects vascular system – indirect cell death
Disulfonated ZnPcS2
Amphophilic
Transported by lipoproteins
Direct cell death
Photochemistry and Photobiology 2001, 74, 656. J. of Nuclear Medicine, 2006, 47, 1119.

29. Photosensitizing Agents: Meta-tetra(hydroxyphenyl)porphyrins (mTHPP)
Commercially available as meta-tetra(hydroxyphenyl)chlorin – (mTHPC)
2nd generation
Improved red light absorption
25-30 times more potent than HpD
More selective toward tumor cells
Most active photosensitizer with low drug and light doses
Not granted approval
Photochemistry and Photobiology 2001, 74, 656. Int. J. Cancer 2001, 93, 720.

30. Photosensitizing Agents: Texaphyrins
Synthetic – porphycene
Water soluble
Related to porphyrins
Absorption between nm (far red)
Sufficiently penetrates tissue
Photochemistry and Photobiology 2001, 74, 656.

31. Photosensitizing Agents: Tin ethyl etiopurpurin
SnET2, Purlytin
Chlorin
Treatment of cutaneous metastatic malignancies
Results of phase III study (934 patients) not yet released
Photochemistry and Photobiology 2001, 74, 656.

32. PDT Trials on Tumor Cells: Breast Cancer
Chest wall recurrences – problem with mastectomy treatment (5-19%)
Study:
7 patients, 57.6 years old (12.6)
89 metastatic nodes treated
11 PDT sessions
Photosensitizing agent: (m-THPC)
meta-tetra(hydroxyphenyl)chlorin
2nd generation photosensitizing agent
Int. J. Cancer 2001, 93, 720.

33. PDT Trials on Tumor Cells: Breast Cancer
Dosage:
Diode laser used to generate l = 652 nm
3 patients
0.10 mg/kg total body weight
48 hr under 5 J/cm2
4 patients
0.15 mg/kg total body weight
96 hr under 10 J/cm2
Int. J. Cancer 2001, 93, 720.

34. PDT Trials on Tumor Cells: Breast Cancer
Results:
Complete response in all 7 patients
Pain – 10 days, Healing – 8-10 weeks
Patients advised to use sun block or clothing to protect skin from light for 2 weeks
4 days after treatment – 1 patient with skin erythema and edema from reading light
6 of 7 patients given medication for pain
Mostly based on size, not lightdose
Recurrences in 2 patients (2 months)
Int. J. Cancer 2001, 93, 720.

35. PDT Trials on Tumor Cells: Skin Cancer
Traditional Treatments:
Surgery, electrodesiccation, cryosurgery, topical application of podophyllin or 5-fluorouracil, radiation
Problems:
High cost, scarring, pigmentation changes, pain, inflammation, irritation
Pharmaceutical Research 2000, 17, 1447.

36. PDT Trials on Tumor Cells: Skin Cancer
Most promising treatment using PDT
Skin highly accessible to light exposure
Most common method
Topical administration of 5-ALA
Non-invasive, short photosensitization period, treat multiple lesions, good cosmetic results, well accepted by patients, no side effects
Pharmaceutical Research 2000, 17, 1447.

37. PDT Trials on Tumor Cells: Skin Cancer
Mechanism of 5-ALA use:
5-ALA formed in vivo in mitochondria by condensation of glycine and succinyl CoA (catalyzed by ALA-syntase)
Subsequent reactions produce protoporphyrin IX (PpIX)
Converted to heme using ferrochelatase and Fe
Heme inhibits synthesis of 5-ALA
Excess administered 5-ALA passes through abnormal epidermis and converts to PpIX
Pharmaceutical Research 2000, 17, 1447.

38. PDT Trials on Tumor Cells: Skin Cancer
Mechanism (continued):
PpIX accumulates with minimized amount of ferrochelatase
Tissues with increased concentration of PpIX undergo phototoxic damage upon light exposure
3PpIX is formed, energy transferred to create 1O2
PpIX nearly completely cleared within 24 hr
Pharmaceutical Research 2000, 17, 1447.

39. PDT Trials on Tumor Cells: Skin Cancer
Clinical Studies performed on superficial skin cancer types:
Actinic keratosis (AK)
Basal cell carcinoma (BCC)
Squamous cell carcinoma (SCC)
Bowen’s disease (BD)
Complete response (CR) – no clinical or histopathologic signs after follow-up
Minimal side effects
Pharmaceutical Research 2000, 17, 1447.

40. PDT Trials on Tumor Cells: Skin Cancer
Pharmaceutical Research 2000, 17, 1447.

41. PDT Trials on Tumor Cells: Skin Cancer
Clinical trials with mono-L-aspartyl chlorin e6 (NPe6)
14 patients – 9 male, 5 female
46-82 years old (64 yrs average)
BCC – 22 lesions, SCC – 13 lesions, papillary carcinoma – 14 lesions
Photodermatol Photoimmunol Photomed 2005, 21, 72.

42. PDT Trials on Tumor Cells: Skin Cancer
Clinical trials (continued)
5 different intravenous doses of NPe6 over 30 minutes (0.5 mg/kg – 3.5 mg/kg)
4-8 hr prior to light administration (due to number of lesions)
Light dose – J/cm2
Argon-pumped tunable dye laser set at 664 nm
Dose dependent on tumor size/shape
Photodermatol Photoimmunol Photomed 2005, 21, 72.

43. PDT Trials on Tumor Cells: Skin Cancer
Photodermatol Photoimmunol Photomed 2005, 21, 72.

44. PDT Trials on Tumor Cells: Skin Cancer
Results:
4 weeks later: 20 of 22 BCC – CR, 18 of 27 other – CR
CR – no evidence of tumor in treatment field
PR – >50% reduction in tumor size
Photosensitivity gone within 1 week (12 of 14)
3 patients – mild to moderate pruritis, facial edema or blistering, erythema, tingling
1 patient – severe intermittent burning pain
1 patient – erythema, edema, moderate pain (gone within 2 weeks)
Photodermatol Photoimmunol Photomed 2005, 21, 72.

45. Conclusions PDT of cancer regulated by: Type of photosensitizing agent
Type of administration
Dose of photosensitizer
Light dose
Fluence rate
O2 availability
Time between administration of photosensitizer and light

46. Conclusions
Tumor cells show some selectivity for photosensitizing agent uptake
Limited damage to surrounding tissues
Less invasive approach
Outpatient procedure
Various application types
Well accepted cosmetic results

47. Conclusions: Clinical Approval of Photosensitizers
Nature 2003, 3, 380.

48. Future Applications: Treatment of Other Diseases
Dermatology
Psoriasis, scleroderma, vitiligo
Rheumatology
Arthritis
Cardiovascular diseases
Artherosclerotic plaque resolution, post-stent implantation
Age-related eye diseases
Macular degeneration
Immunotherapy
Nature 2003, 3, 380. Photochemistry and Photobiology 2001, 74, 656.

49. Future Applications: Tumor Detection Using Fluorescence
Mechanism by which HpD selectively accumulates in tumor cells – not well understood
High vascular permeability of agents?
Testing photosensitizing agents:
Porphyrins, haematoporphyrins, HpD, ALA-D
Administer photosensitizer and monitor fluorescence with endoscope
SCC shows increased fluorescence
More invasive tumors show even greater fluorescence
Nature 2003, 3, 380.

50. Future Applications: Tumor Detection Using Fluorescence
a: Green vascular endothelial cells of a tumor
b: Red photosensitizing agent localizes to vascular endothelial cells after intravenous injection
Nature 2003, 3, 380.

51. Future Applications: Photosensitizing Drugs
Improved Specificity and Potency
Better photosensitizers developed and under investigation in clinical trials
Use of carriers – conjugated antibodies directed to tumor-associated antigens
New compounds that absorb light of longer wavelength – better tissue penetration
New compounds with less skin photosensitivity
Improved Efficacy
Creating a preferred treatment of cancer
Nature 2003, 3, 380.

52. Thank you