anti-tumoraler effekt von artesunate thomas efferth ... · anti-tumoraler effekt von artesunate...
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Anti-tumoraler Effekt von ArtesunateThomas Efferth
Johannes Gutenberg-Universtät Mainz
$
TCM im Westen
€ $€ $€ $€ $€ $€
Alternativmedizin und Esoterik
Exportschlager2004: 98 Mio $2009: 160 Mio $
TCM in Fernost
TCM ersetzt die fehlende medizinische Grundversorgung in
unterentwickelten ländlichen Regionen Chinas
• Gesundheit:• Yin und Yang im Gleichgewicht • Ausgewogenheit von Körper, Geist und Seele
• Krankheit:• Disharmonie von Yin und Yang• Energiefluss von Qi in den Meridianen ist gestört
• löst Krankheiten aus
Begriffs-definitionen
Physiologische Funktionensekundärer Pflanzenmetaboliten
1. Schutz vor Parasiten (Insekten, Würmer etc.)
2. Schutz vor mikrobiellen Infektionen(Viren, Bakterien, Pilze, etc.)
3. Schutz vor Herbivoren4. Anlocken von Bestäubern (Insekten)
Vinca rosea (Catharanthus roseus)
B, biological drugs: peptides or proteins (> 45 residues)N, natural productND, derived from natural product, semi-synthetic modificationS, totally synthetic drugS*, synthetic with pharmacophore from natural product V, vaccineNM, natural product mimic
Newman and Cragg, J Nat Prod (2007)
Krebsmedikamente (1940 – 2006)
Traditionelle Chinesische Medizin: Anti-Tumor Screening
250.000 Pflanzen weltweit
Medizinalpflanzen
TCM
Medizinalmarkt Kunming
Medizinalpflanzen
Bioaktivitätsgeleitete Isolation von PflanzeninhaltstoffenPhytochemie
Molekulare PharmakologiePharmakogenomik,
Validierung von Zielstrukturen mit Transfektanten und knock outsIn vivo Validierung
Strategie
Bioactivity-Guided Isolation of Natural Products
010
20304050
60708090
Petrolether Ethylacetat Methanol Wasser
Zellw
achs
tum
(% d
er K
ontro
lle)
Quisqualis indica L.
Efferth et al., Mol Cancer Ther (2008)
1 2 3Extract Testing
andFractionation
Structure Elucidation
0
20
40
60
80
100
120ce
ll gr
owth
of
leuk
emia
cel
ls(%
of
cont
rol a
ssay
)
0
2
4
6
8
10
12
14
16
18
20
inhi
bitio
n ra
dius
[mm
] in
the
antif
unga
l ass
ay
256 extracts tested
Efferth et al., Mol Cancer Ther (2008)
• Artemisia annua L. (Compositae) • Caesalpinia sappan L. (Caesalpiniaceae)• Curcuma longa L. (Zingiberaceae)• Eleutherococcus senticosis Maxim (Araliaceae)• Hydnocarpus anthelmintica Gaertner (Flacourtiaceae)• Lonica japonica Thumb. (Caprifoliaceae)• Quisqualis indica L. (Combretaceae)• Salvia miltiorrhiza Bunge (Lamiaceae)
Anticancer Screening of TCM Plants
5 9 1 1 4 4 3 1 0 02 4 2 6 2 2 5 37 4 9 38 1 8 3 5 0 6 1 5 9 6 94 0 7 97 8 2 3 2 9 5 81 3 9 2 88 5 9 68 6 9 1 1 5 7 66 7 8 8 8 0 1 1 1 1 3 3 8 5 13 3 6 55 71 1 0 3 1 6 04 2 7 2 4 68 2 1 1 31 41 92 7 4 77 3 5 4 7 01 1 29 07 1 9 4 1 1 66 3 76 8 8 7 1 1 54 8 39 8 9 5 1 2 5 51 8 4 9 1 0 7 7 1 1 83 2 3 62 2 1 1 1 7 1 1 9 23 0 6 64 5 1 2 6 1 2 76 2 6 4 1 0 4 1 0 51 0 81 6 1 0 71 7 8 9 1 1 9 5 27 51 2 0 1 2 1 1 2 24 1 1 0 9 8 4 1 0 31 0 65 3 4 9 2 52 0 4 5 6 4 4 1 0 2 9 9 1 0 17 6 3 8 1 2 4 3 5 9 7 1 2 51 2 3
57
58
41
54
1
4
26
28
45
50
9
52
8
13
19
21
49
30
32
43
47
33
35
17
44
46
20
22
7
37
2
3
36
31
29
24
59
23
40
16
18
27
56
38
12
15
11
53
60
39
42
51
34
5
10
55
14
6
48
25A B C1
2
3
< - 0.9- 0.9 to - 0.7- 0.7 to -0.5- 0.5 to - 0.3- 0.3 to - 0.1- 0.1 to 0.1 0.1 to 0.3 0.3 to 0.5 0.5 to 0.7 0.7 to 0.9 > 0.91 (resistant)
2 (sensitive)
3 (sensitive)
A B C
Cluster 1 Cluster 2 Cluster 3 2 Testsensitive 5 17 6resistant 18 9 0 P=3.71 x 10-4
Microarray and Cluster Analysis
Efferth, Curr Drug Targets (2006)
cell lines:genes:
resistancegenes
sensitivity genes
color code:lowexpression
highexpression
Activity of ArtesunateTowards Transfected Cell Lines
-GCSCDC25A
resistance genesensitivity gene
Efferth, Mol Pharmacol (2003) [email protected]
Li et al., Cancer Research (2008)
Induction of DNA Strand Breaks by Artesunate
A. Single Cell Gel Electrophoresis C. Immunofluorescence of -H2AX Foci
B. Westernblot of -H2AX
D. Quantification of -H2AX Foci
Identification of Artemisinin-Binding Proteins in silico and in vitro
[email protected] et al., Molecular Biosystems (2012)
Binding of Artemisinin to TCTP(Translationally-Controlled Tumor Protein)
residues 23-50 and 112-138
Eichhorn et al., , Biochemical Pharmacology (2013)
e. Transfection of BCL-2
00,20,40,60,8
1
0 1 2 3 4Art esunate (µM)
WEHI7.2Hb12
Transfectionwith Bcl-2
Artesunate (µM)Re
lativ
e A
bsor
banc
e
Efferth et al., Int J Oncol (2002)Efferth et al., PLoS One (2007)
A0
G0G1
G2MS
Artesunate Induces the Intrinsic Pathway of Apoptosis
Dell‘Eva et al., Biochem Pharmacol (2004)
Artesunate Inhibits Angiogenesis and Tumor Growth in vivo
untreated control artesunate treatment
Matrigel Plug Assay
• Angiostatin• Collagens 18A1 and 4A2• Fibronectin 1• Hypoxia-inducible factor 1• Matrix metalloproteins 9 and 11• Nitric oxide synthase 2A• Plasminogen activator (urokinase)• Thrombospondin• Tissue factor (thromboplastin) • Vascular endothelial factor C
Microarray Analysis Xenograft Nude Mice Model
Anfosso et al., Pharmacogenomics J (2006)
Screening (n = 10)
Enrolment (n = 10)
Treatment I7 days - 100 mg/day21 days - 200mg/day
Tumor responseBiomarkers, tumor size (n
=10)
Clin
ica
l fol
low
up
Day 0
Day 28
Clin. relapse(n = 6)
Symptom-free, alive (n = 6)
Treatment II28 days -
200mg/day
Mortality(n= 2)
Clinical Phase I/II Trial:Activity of Artesunate in Cervix Carcinoma
- Open label, single centerpilot study
- Service de Cancerologie,CHU Treichville, Abidjan, Côte d’Ivoire
- Ten women with cervicalcarcinoma
(stage: 1 x IIIa, 4 x IIIb, 4 x IVa, 1 x IVb)
Mortality(n= 2)
[email protected] et al, Anticancer Res. (2011)
10987654321
Response: 10 x remission.Median time for the disappearance of vaginal blood loss, watery discharge and pain = 7 days (min 3, max 21).
Adverse effects: Grade 1 or 2: 5 patients with “flu-like syndrome”, headache and abdominal painNo grade 3 or 4 adverse events
Average time for clinical relapse: 6 months (min 4, max 8)4 refractory tumors treated again: 2 patients died, 2 in remission
Survival time : 12 months (min 8, max 13), (expected: 4)
Clinical Phase I/II Trial:Activity of Artesunate in Cervix Carcinoma
Jansen et al, Anticancer Res. (2011)
(TUNEL-Assay (Apoptosis):Activity of Artesunate in Cervix Carcinoma
0
10
20
30
40
timepoint 1
timepoint 2
timepoint 3
Tumor 643
Jansen et al, Anticancer Res. (2011)
A. p53 B. EGFR C. Ki-67
D. CD71 (TfR) E. CD31 F. Quantification
0
20
40
60
80
100
120
140
160
CD31
Num
ber o
f Blo
od V
esse
ls
0
10
20
30
40
50
60
70
p53 EGFR Ki-67 CD71
% P
ositi
ve C
ells
time point 1time point 2time point 3
Immunohistochemical Biomarkers:Activity of Artesunate in Cervix Carcinoma
[email protected] et al, Anticancer Res. (2011)
Zusammenfassung
1. Die Grundlagen der TCMJahrtausendealte Geschichte und Philosophie
2. Molekulare PharmakologiePhytochemie von WirkstoffenMolekularbiologische Aufklärung der Wirkmechanismen
3. Klinische StudienNachweis der Wirksamkeit in Tumorpatienen
Vielen Dank für Ihre Aufmerksamkeit!