Photodynamische Therapie

(1)

Photodynamische Therapie (PDT) und

Photodynamische Diagnose (PDD)

Großes Forschungsgebiet

Klinisches Versuchsstadium mit Übergang zur klinischen Routine

(2)

Krebserkrankung

alle Körperzellen haben nur eine begrenzte Lebensdauer. Nach einer bestimmten Zeit wird der programmierte Zelltod (Apoptose) aktiviert.

Bei der Chemotherapie werden Zellgifte verabreicht, die die Tumorzellen stärker schädigen als die gesunden Zellen.

Da aber auch gesunde Zellen geschädigt werden, gibt es eine Vielzahl von schweren Nebenwirkungen.

Wenn diese entarteten Zelle vom Immunsystem nicht erkannt und vernichtet werden, entsteht Krebs.

Aber: Zellen können entarten und „vergessen“ zu sterben.

Ewige Jugend ist nicht möglich, denn

(3)

1. Grundlagen der Photodynamischen Diagnose und Therapie

(4)

Grundidee der

Photodynamischen Therapie

Es werden Farbstoffe („Photosensibilisatoren“) in den Körper

gegeben, die nicht giftig sind und die sich in dem erkrankten Gewebe selektiv anreichern.

Wenn ein ausreichender Anreicherungsunterschied zum gesunden Gewebe erreicht ist, werden über Laserlicht

• phototoxische Prozesse ausgelöst (Therapie) oder

• die erhaltene Fluoreszenz beobachtet (Diagnose).

(5)

Gesundes Gewebe Tumor Gewebe

Prinzip von PDT

(6)

Verabreichung des

Photosensibilisators (PS)

Topisch oder systemisch

Wichtig: Dichte des PS und Einwirkzeit

(7)

Zeitliche Entwicklung der Dichte des PS

(8)

(9)

(10)

Bestrahlung mit blauem Licht

Tumor Zellen emittieren rote Fluoreszenz

Photodynamische Diagnose (PDD)

(11)

Bestrahlung mit rotem Licht

Licht und PS generieren phototoxische Stoffe

Tumor Zellen werden abgetötet Photodynamische Therapie (PDT)

(12)

(13)

(14)

(15)

(16)

(17)

Singulett-Sauerstoff-Quantenausbeute

  ₂

et T

T    k  O





 _

_T = Triplettquantenausbeute

_T = Triplettlebensdauer

k_et = Ratenkonstante für den Energietransfer Sens(T₁) + ³O₂  Sens(S₀) + ¹O₂ [O₂] = Konzentration von O₂

(18)

Beispiel: Dermatologie: Rumpfhautbasaliom

Vor Therapie

PDD

5 Wochen nach Therapie

5 Wochen nach weiterer Therapie

Krutmann, Hönigsmann: Handbuch der dermatologischen Phototherapie und Photodiagnostik

(19)

Zungengrund-Karzinom

H. Stepp, München, Großhadern

(20)

Barrett-Syndrom

Ell, Großer, Wiesbaden

(21)

H. Stepp, München, Großhadern

(22)

(23)

Patient mit Hauttumor Nach Therapie

Unmittelbar nach der PDT hatte er (gegen den ärztlichen Rat)

eine Bergwanderung vorgenommen Photofrin führt zu einer

14-tägigen Lichtempfindlichkeit.

Bei ALA ist nach 24 Stdn. wieder der Normalzustand erreicht

(24)

Glyome

Tumor leuchtet durch die Hirnhaut Fluoreszenz hilft beim millimeter- genauen Abtrag des Tumors

„Quetsch-Präparat“

Verwendeter Sensibilisator:

ALA in Orangensaft gemischt, 4 Stdn. vor Operation getrunken

(25)

Senile Macula-Degeneration

(26)

(27)

(28)

So sieht es der Patient:

(29)

H. van den Bergh, Lausanne

vor nach der Therapie

(30)

Therapie: Familiäre adenomatöse Polyposis

•Polypen (Adenome) im Dickdarm

breiten sich aus

•Meist gutartig –

können aber entarten

•Ausbreitung über ganzen Darmbereich

(31)

Vor Therapie Nach Therapie

(32)

ERCP - Endoskopisch-retrograde

Cholangio-Pankreaticographie

(33)

Gallengangskarzinome

Vor PDT Nach PDT

•Stenosen gehen im

Idealfall komplett zurück

Verbesserung der

Lebensqualität, aber nur palliativ

Forschungsbedarf:

•Ungeklärte Fälle ohne erfolgreiche Therapie

•Anreicherungskinetik mit neuen PS

(34)

Patient bei Photodynamischer Therapie des Larynx

(35)

Perfusionsmodell

Dr. Linder, Lungenklinik Hemer

(36)

Schnitte an Lungengewebe

Neues Projekt gefördert durch Deutsche Krebshilfe:

Physikalische und medizinische Grundlagen zur

Photodynamischen Therapie peripherer Lungenkarzinome

Kooperation mit Westpfalz-Klinikum und Lungenklinik Hemer

(37)

(38)

Photosensitisers (PS)

A long list of various sensitisers has been developed, tested and applied in medicine

See for example:

Mark Wainwright (Liverpool):

Photosensitisers in Biomedicine, Willey-Blackwell 2009

(39)

Einteilung der PS

 Typ 1 Mechanismus:

PS wird direkt zu einem reaktiven Stoff

 Typ 2 Mechanismus:

Bildung von Singulett-Sauerstoff

(40)

Einteilung der PS

 1-te Generation: HPD

Hämotoporpyrinderivat (Photofrin, Photosan)

 2-te Generation:

Phorbide (Chlorine), Benzeoporphrin-Derivate, Purpurine, Bakteriochlorophyll, Phtalocyanine, Naphthalocyanin

 3-te Generation: Delta-Aminolävulinsäure ist „nur“ eine Vorstufe von PS

(41)

Photosensitisers

• Here discussed in detail:

• Haemotophorhyrine Derivates

• Aminolaevulinic Acid (ALA)

• Methyl ester of ALA

• Hexyl ester of ALA

• Chlorin e6

(42)

Porphyrin Haem Chlorin e₆

(43)

(44)

ALA or MAL-induced PPIX. Schematic illustrating the interaction of the heme biosynthesis pathway with exogenous ALA or MAL to give intracellular PPIX.

Abbreviations are ALA-D = ALA dehydratase; ALA-S = ALA synthetase; Coprogen III = coproporphyrinogen III;

CPO = coproporphyrinogen oxidase; FCH = ferrochelatase; HMB = hydroxymethylbilane,

PBG-D = porphobilinogren deaminase; protogen III = protoporphyrinogen; PPO = protoporphyrinogen oxidase;

Urogen III = uroporphyrinogen III; UCS = uroporphyrinogen cosynthase, UGD = uroporphyrinogen decarboxylase.

(45)

Esters of ALA to improve membrane penetration

(46)

Why inactivation of bacteria by photodynamic therapy?

Increasing problems with resistant

bacteria

(47)

Antibiotic Resistance of Bacteria

Reasons for increasing antibiotic resistance:

• Inappropriate application of antibiotics

• Failure to complete treatment

• Widespread use in livestock feedstuff

The worldwide growth of multi-drug

resistant bacteria makes it neccessary to to find alternative antibacterial

therapeutics to which bacteria will not be easily able to develop resistance.

(48)

Mechanisms for

photodynamic inactivation of bacteria

I  gram positive bacteria

Direct translocation of the PS to plasma membrane

II gram negative bacteria

Initial increase in the permeability of the outer wall continued by translocation of the PS to the inner plasma membrane

Furthermore

intercalation of PS into double stranded DNA  breaks in both single and double stranded DNA and loss of supercoiling

 Generation of reactive cytotoxic species after photoactivation

(49)

(50)

(51)

Characteristic cell wall of Mycobacteria

•unusual thick hydrophobic cell wall

•consisting of peptidoglycan, arabino-galactan and mycolic-acids

 barrier preventing diffusion of hydrophilic

and hydrophobic compounds into the cell

(52)

  Actinomycetes

white light (10xmagn) blue light after ALA-application

Starting situation

(53)

Bacteria  Actinobacteria 

Actinobacteridae  Actinomycetales  Corynebacterineae 

Mycobacteriaceae  Mycobacterium

 Mycobacterium tuberculosis ???????

Phylogenetics

(54)

Multiresistant Bacteria – a big problem in medicine

Generation of new mutants of many bacteria strains

 methillicin-resistant Staphylococcus aureus

 Vanomycin-resistant Enterococci

 MDR-Mycobacterium tuberculosis

Contagious disease caused by the bacterium M.tuberculosis, which affects lungs, the central nervous system, lymphatic system,

circulatory system, genitourinary system and bone.

Cavern in a infected lung caused by Tuberculosis

(55)

The strain Mycobacterium

• Gram-positive acid-fast bacterium

• 1µm -10 µm long

• aerobic

• nonmotile

• rod shaped

• division between slowly and rapidly growing

• pigmentation

Mycobacterium tuberculosis

Other common representatives : M. leprae, M. paratuberculosis, M.bovis, M.marinum

(56)

Conventional therapy of Tuberculosis

• Diagnosis:

• radiology,

• tuberculin skin test

• serological test

• microbiological smears and cultures

• Treatment:

• Isoniazid (INH)

• Rifampicin (RIF)

• Ethambutol

• Pyrazinamide

New perspective:

Photodynamic Diagnosis and

Inactivation

(57)

Model organisms of M. tuberculosis

•Mycobacterium phlei AF480603/DSM 43239, ATCC 11758 (S1) (University of Swansea, Institute of Life Sciences School of Medicine Singleton Park SA2 8PP Swansea, Wales UK)

•Mycobacterium smegmatis mc2155 (S2 organism)

(German Collection of Microorganisms and Cell Cultures DSMZ)

(58)

Pharmacokinetic of ALA induced Porphyrins

Fluorescence maxima after ALA and administration

Emission peaks in different bacteria strains after sensitation and irradiation at 410 nm

300 400 500 600 700 800 900 1000 1100

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4

first peak at 619nm second peak at 679nm Autofluorescence at 531nm

M.smegmatis 6mM ALA

Intensity [a.u.]

wavelength [nm]

0min 60min 120min 180min 240min 300min

Normalization of the total fluorescence to the autofluorescence peak of the bacterial suspension

Bacterial strain Photosensitizer/

precursor

Emission peak [nm]

E.coli m-ALA 615

Corynebacterium glutanicum h-ALA None

Mycobacterium phlei ALA 615

Mycobacterium phlei h-ALA 618

Mycobacterium phlei Photosan 630

Mycobacterium phlei Photofrin 633

Mycobacterium smegmatis ALA 621

Mycobacterium smegmatis h-ALA 619

Mycobacterium smegmatis Photosan 630

Mycobacterium smegmatis Photofrin 635

Streptomyces coelicolor ALA -

Streptomyces coelicolor m-ALA 616

Streptomyces coelicolor h-ALA -

(59)

PS localisation in bacteria

Setup of fluorescence microscopy for two-dimensial spatially resolved spectroscopy

M. smegmatis after ALA-sensitization under blue light illumination.

B: 3D-plot of the fluorescence intensity at 619 nm

(60)

Lens

CCD Camera Beam Splitter

Interferogram

Fourier Analysis Spectrum

Interferometer SpectraCube

^R

(61)

Emission Spectra of Porphyrins

Emissions Spectra of Porphyrins under blue light illumination; Substances dissolved in DMSO

400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 0

50 100 150 200 250 300 350 400

Intensity [a.u.]

wavelength [nm]

Typical PpIX doublepeak, known from tumortherapy

(62)

Pharmacokinetic of ALA induced Poprhyrins

-200 0 200 400 600 800 1000 1200 1400 1600

-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

Intensity [a.u.]

Time [min]

M.phlei 6mM ALA M.phlei 6mM hALA M.phlei negativ

-200 0 200 400 600 800 1000 1200 1400 1600

-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0

Intensity [a.u.]

Time [min]

M.smegmatis 6mM ALA M.smegmatis 6mM hALA M.smegmatis negativ

Time-course of porphyrin synthesis in M.phlei and M.smegmatis over a period of 24h; fluorescence-peaks at 620nm dependet of the time

Mycobacterium strains enrich porphyrins after ALA and h-ALA administration detected by fluorescence peaks at about 620 nm

By HPLC (High Performance Liquid Chromatography) analyses the major porphyrin could be identified as coproporphyrin.

(63)

Photosensitizer

Precursor Aminulevulinic Acid

Heme biosynthesis pathway (E.Malitz, 2002)

Human Cells

Myco-

bacteria

(64)

Photosensitizer: Chlorin e ₆

400 500 600 700 800 900 1000 1100

0 5000 10000 15000 20000

Intensity

Wavelength [nm]

(65)

Pharmacokinetic of Chlorin e ₆

0 5 10 15 20 25

1 2 3 4 5 6 7 8 9 10

Intensity [a.u.]

time [h]

0,05 mM 0,1 mM 0,3 mM

0 5 10 15 20 25

1 2 3 4 5 6 7 8 9 10

Intensity [a.u.]

time [h]

0,05 mM 0,1 mM 0,3 mM

•Pharmacokinetik of Chlorin e6 in Mycobacteria, excitation at 410 nm

•Significant uptake of the PS in the cells or bound to the cell membrane

(66)

Technical data for irradiation

Diode LASER 1

•λ = 662 ± 3 nm

•E = 140 ± 18 J/cm²

•Diode LASER 2

•λ = 630 ± 10 nm

•E = 16, 50, 160, 320 J/cm²

Treatment of bacteria:

Dilutions: 0, 10^-1, 10^-2 Sensitization with PS after Incubation time Wash twice in PBS

Irradiation for 20 minutes survival rate by

colony forming units (CFU)

(67)

PDI ALA M.phlei

0 16 48 161 322

0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7

3.0 M.phlei 16 h ALA

survival fraction [N/N0]

energy density [J/cm²]

unmoved stirred aerated

0 16 48 161 322

0.5 1.0 1.5 2.0 2.5

3.0 M.phlei 42 h ALA

 mortality rate up to 80%

 best results by using an energy density of 160 J/cm² and 320 J/cm²

(68)

PDI ALA M.smegmatis

0 16 48 161 322

0.5 1.0 1.5

2.0 M.smegmatis 16 h ALA

0 16 48 161 322

0.5 1.0 1.5

2.0 M.smegmatis 42 h ALA

 best results after 42h ALA treatment

(69)

PDI by using Chlorin e ₆

0 0.23 0.68 0.9 1.8 2.7

0.5 1.0 1.5 2.0 2.5

M.phlei

concentration [mM]

0 0.23 0.68 0.9 1.8 2.7

0.5 1.0 1.5 2.0

2.5 M.smegmatis

concentration [mM]

 best results by using the combination of 0,9mM Chlorin e6 and gassing

(70)

TEM of Chlorin e

₆

treated M.smegmatis

 membrane lysis in all irradiated cells

 lost of cell structure in all treated bacteria

before irradiation after irradiation

(71)

Conclusions Concerning Mycobacteria

 Both Mycobacteria strains enrich porphyrins after ALA and hALA administration detected by fluorescence peaks at about 620 nm

 Alternative method for diagnosis of vital Mycobacteria in infected lessons (esp. Leprosy)

 Successful inactivation of M.phlei and M.smegmatis using ALA and Chlorin e6

 Major porphyrin in M.phlei and M.smegmatis could be identified as coproporphyrin by using HPLC analyses*

*High-Performance Liquid Chromatography

(72)

Are there already clinical applications of aPDT?

Yes!

For example in dentistry

and against Acne Vulgaris

(73)

Several Systems for Dentistry

• Ondine Biopharma (www.ondinebiopharma.com)

in North America is using methylene blue (MB) and 660-nm light for treating eriodontitis (and nasal MRSA decontamination)

• HELBO Photodynamic Systems (www.helbo.at) in Austria is using toluidine blue O (TBO) and 635-nm light to treat

periodontitis and endodontic infection and

• Denfotex (www.denfotex.com) in UK also uses TBO and 635-nm light to treat endodontics, periodontitis and caries.

(74)

Antimicrobial Photodynamic Therapy (aPDT) Clinical Approach for Parodontitis/Perimplantitis

Start situation: Inflammation, STI> 4 mm, swelling, pain

Professional Cleaning

Pathogen bacteria are left

Application of blue dye

Staining of microorganism,

1-3 min Flushing with water Irradiationfor 1 min per 1 cm² tooth surface

Destruction of bacteria

(75)

(76)

Helbo

• Website: Bacteria reduction of 99 %

10⁹ bacteria, reduction 99 %  10 Million survivors

(77)

Fibre applicator

(78)

Raytracing (ASAP, Breault)

(79)

(80)

(81)

Recently published Review Article:

(82)

Michael R. Hamblin and coworkers

Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA

In-vivo Study

(83)

Dai et al.

(84)

Dai et al.

(85)

Summary

Advantages of antimicrobial PDT:

 Repeatable

 High efficiency*

 No resistance

*Heyke Diddens (Biophysics, Lübeck, Germany):

Efficiency: 10⁹

Combination of antiseptics and aPDT is much more efficient than each of the single approachs

(86)

Outlook

Dai et al.

(87)

Membrane Structure of Fungus

Dai et al.

(88)

Expected Development

Dai et al.

(89)

Problem Pathogens in the Hospital Setting

W. Conrad Liles, MD, PhD

Professor and Vice-Chair of Medicine Director, Division of Infectious Diseases

Canada Research Chair in Infectious Diseases and Inflammation

University of Toronto

Presented in Quebec Thursday Sept. 23 2010

(90)

Clin Infect Dis 2010;50:1081

Nature Medicine 2010; 16:628

Methicillin-resistant Staphylococcus aureus (MRSA)

(91)

IDSA: Infectious Diseases Society of America

(92)

(93)

Looking ahead…

The future of antimicrobial

development

(94)

Approval of New Systemic Antibacterial Agents

Clin Infect Dis 2009; 48:1

(95)

Approval of New Systemic Antibacterial Agents

Clin Infect Dis 2009; 48:1

(96)

Drugs in Development

European Medicines Agency, European Centre for Disease Prevention and Control. 2009.

http://ecdc.europa.eu/en/publications/Publications/0909_TER_The_Bacterial_Challenge_Time_to_React.pdf.

(97)

Barriers to Drug

Development

1. Limited potential financial gain for developers

– Niche market

– Profits limited in first years after release – Curative

– Lifespan of newly released ABx can be short

Torres C. Nat Med 2010; 16: 628-631.

IDSA. http://www.idsociety.org/10x20.htm. Accessed August 14, 2010.

Morel CM et al. BMJ 2010; 340: c2115.

(98)

Barriers to Drug

Development

2. Study expectations and regulations

– Patients are non-uniform, difficult to accrue – No defined standard for NI endpoints

– Shift towards superiority trials

– Fully resistant pathogens can’t be studied in an RCT

Torres C. Nat Med 2010; 16: 628-631.

IDSA. http://www.idsociety.org/10x20.htm. Accessed August 14, 2010.

Morel CM et al. BMJ 2010; 340: c2115.

(99)

Photodynamische Therapie