Download RGD Tumor Targeting Conjugates - MSU Chemistry

Arg-Gly-Asp (RGD) Tumor
Targeting Conjugates
Ruth Njeri Muchiri
Michigan State University
Department of Chemistry
Cancer
Uncontrolled cell growth, invasion and metastases
Normal cell division
DNA damage
( No repair)
Cell suicide (apoptosis)
Cancer cell division
Damaged cell
Tumor
http://www.cancer.gov/
Causes & Types of Cancer
Main Causes
 Radiation
 Age-related causes
 Chemicals
Types
 Solid cancers
Examples: lung, breast, bowel cancer
 Non-solid cancers
Examples: leukemia, lymphoma, myeloma
http://www.immunotherapyforcancer.info
Cost of Cancer
2007
11.7 million
USA population with
cancer
2008
$895 billion
(1.5% World’s GDP)
World’s economic loss
2010
1,529,560
569,490
New cancer cases in USA
New deaths in USA
http://www.cancer.org/research/cancerfactsfigures/cancerfactsfigures/cancer-facts-and-figures-2010
Attempts Towards Cancer Treatment
Surgery
Chemotherapy
Radiotherapy
Agent
Examples
Alkylating agents
Cyclophosphamide, Oxaliplatin, Carboplatin, Chlorambucil,
Mechloethamine, Melphalan
Antimetabolite agents
.
Folate antagonists, Methotrexate, Fludarabine, Cytarabine
Plant alkaloids and
terpenoids
Doxorubicin, Vinca alkaloids, Taxanes, Camptothecin
Antitumor antibiotics
Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin,
Mitoxantrone
http://www.immunotherapyforcancer.info
http://www.medindia.net/index.htm
Conventional Chemotherapy Treatment
Pros
Cons
 Less mutilating
 Non specific
 Neo-adjuvant treatment
 Toxic
 Preferred in metastasis
cases
 Multiple drug resistance
http://www.cancer.gov/
Development on Chemotherapy
One leg good, two legs better: Cocktail therapy
VAC (Etoposide, Doxorubicin, Cyclophosphamide)
CVM (Cisplatin, Vincristine, Methotrexate)
MCP (Mitomycin-C, CCNU, Procarbazine)
100
Patients with
extensive lung tumor
90
80
High toxicity observed
% Survival
70
Patients with limited
lung tumor
60
50
40
30
Bevacizumab + Gemcitabine
in advanced pancreatic cancer
20
10
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Time (months)
Pavlidis, A. N.; Tolis, T. F.; Briassoulis, E. C.; Sowla, A.; Klouvas, G. D. Lung Cancer 1991, 1, 279-283.
Kindler, H. L. et al. J. Clin. Oncol. 2005, 23, 8033-8040.
Development on Chemotherapy
Drug analogues
Improved binding but low specificity
Rapoport, N. Y.; Herron, J. N.; Pitt, W. G.; Pitina, L. J. Controlled Release 1999, 58, 153–162.
Bigioni, M.; Salvatorea, C. Biochemical Pharmacology 2001, 62, 63–70.
Targeted Cancer Therapy
Specific
Reduced toxicity
Maximization of drug effect
Picture from: http://www.isncc.org/files/100-Wed_Ball_1-3_0830.2_Ouwerkerk.pdf.
Tumor Targeting Mechanism
Cancer Cell
Ojima, I. Acc. Chem. Res. 2008, 41,108–119.
Tumor Targeting Molecules
Ligand
Target
Target location
Diverse
Diverse
CD44
Layilin
HARE
CDC37
Epithelial
Ovarian
Colon
Stomach
Antibodies
Hyaluronic acid
Somastatin analogues
c(Arg-Gly-Asp-x-y)
SSTR1-5
αvβ3 integrin
Gastro-intestinal cells
Endothelial cells
Epithelial
Glioblastoma cells
Jaracz, S.; Chen, J.; Kuznetsova, L. V.; Ojima, I. Bioorg. Chem. 2005, 13, 5043-5054.
Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem. Int. Ed. 1997, 36, 1374-1389.
Outline
 RGD definition
 RGD–integrin receptors interaction
 Cilengitide® (Merck)
 RGD conjugate design and effect on activity
 Future directions
Arg-Gly-Asp (RGD)
Natural RGD containing proteins
G
R
D
Proteins
Sequence
Fibronectin (450 kDa)
AVTGRGDSPASSK
Fibrinogen α-chain
(72 kDa)
TSYNRGDSTFESK
λ-receptor on E.coli
(47.7 kDa)
GSFGRGDSDEWTF
Sindbis coat protein
(15.5 kDa)
GVGGRGDSGRPIM
α-lytic protease
ACMGRGDSGGSWI
Testis specific basic
protein
KSRKRGDSADRNY
Pierschbacher, M. D.; Ruoslahti. Nature 1984, 309, 30-33
RGD Receptor- αvβ3 Integrin
Function
Control angiogenesis,
cell proliferation & migration
PDB1JV2.
αv
β3
RGD-peptide
Xiong, J. P. et al. Science 2002, 296,151-155.
Mn2+
Cyclic RGD
Arg-Gly-Asp-f-(N-Me)Val
RGDxy
1FNF
Natural peptide – Fibronectin (450 kDa)
AVTGRGDSPASSK
Leahy, D. J.; Aukhil, I.; Erickson, H.P. Cell 1996, 84, 155–164.
Peishoff, E. C. et al. J. Med. Chem. 1992, 35, 3962-3969.
RGD - αvβ3 Integrin Interaction
Mn2+
H-bonding
Metal ion co-ordination
PDB1JV2
Xiong, J. P. et al. Science 2002, 296,151-155.
Advantages of RGD
 Selective binding - ligand for tumor marker, αvβ3 integrin
 Has better cellular uptake
 Easy to synthesize - solid phase synthesis
 Application in tumor imaging
Aguzzi, M.S. et al. Blood, 2004, 103, 4180-4187..
Liu, S. Bioconjugate Chem. 2009, 20, 2199-2213.
Advantages of RGD
Liu, S. Bioconjugate Chem. 2009, 20, 2199-2213.
Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25, 1637–1638.
Outline
 RGD definition
 RGD –integrin receptors interaction
 Cilengitide®
 RGD conjugate design and effect on activity
 Future directions
Cilengitide®
Phase III clinical trial (Merck Germany)
Active against glioblastoma (brain
tumor)
Induce apoptosis in tumors cells
http://www.chemblink.com/products/188968-51-6.htm
Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25, 1637–1638.
Cilengitide® Mode of Action
 Inhibit angiogenesis
Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. Acc. Chem. Res. 2009, 42, 969-980.
Stupp, R.; Ruegg, C. J. Clin Oncol. 2007, 25, 1637–1638.
Proof of Principle
In vivo Studies
Blood vessels in brain tumor
Brain tumor cells inoculated in
nude mice
E1 - Control
E2 - After Cilengitide treatment
A
Brain tumor
B
Phase II clinical trials
Radiographic response to
Cilengitide in glioblastoma patient
A. Before treatment
B. After treatment
Tagai, T. et al. Int. J. Cancer 2002, 98, 690-697.
Reardon, A. D. et al. J. Clin. Oncol. 2008, 26, 5610-5617.
cRGD Conjugates:Old Chemotherapy in New Form
Pozzo, A. D.et al. Bioorg & Med. Chem. 2010, 18, 64–72.
cRGD Conjugates
Cilengitide modification
Vachutinsky, Y. et al. J. Control. Release 2010, doi:10.1016/j.jconrel.2010.02.002.
Outline
 RGD definition
 RGD –integrin receptors interaction
 Cilengitide
 RGD conjugate design and effect on activity
 Future directions
Effect of RGD Conjugate Design on Activity
Multimerization
Immobilization
Sites of RGD
Linker Bond
cRGD Peptide Immobilization Sites
PCLA –(RGD)-PEG- PCLA
PCLA -PEG- PCLA
RGD-PCLA -PEG- PCLA-RGD
PCLA: poly(ε-caprolactone-co-lactide)
PEG: poly(ethylene glycol)
Approved by FDA
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
Liu, C. B.; et al. J. Biomed. Mater. Res. B Appl. Biomater. 2007, 165–175.
cRGD Immobilization on Hydrophobic Site
n=6 to 32
x=3 to 7
y=4 to 9
PCLA-PEG-PCLA
RGD-PCLA-PEG-PCLA-RGD
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
cRGD Immobilization on Hydrophilic Site
PCLA-PEG-PCLA
PCLA-(RGD)-PEG-PCLA
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
cRGD Peptide Immobilization Sites
RGD–cell interaction
PEG blocks
PCLA blocks
RGD peptides
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
Effect of cRGD Immobilization Sites
30
Cell adhesion (%)
25
20
15
10
5
Thiazolyl blue assay
Cell line used: Chondrocytes +
fetal bovine serum
0
RGD(-PEG)
RGD(-PCLA)
Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Biomaterials 2010, 31, 7873-7882.
Unmodified
No RGD
cRGD Conjugate Design and Effect on Activity
Immobilization
of RGD Sites
Multimerization
Hydrophilic site
Enhance RGDintegrin interaction
Linker Bond
cRGD Conjugate Multimerization
Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168 – 2181.
Effective cRGD Conjugate Multimerization
Antitumor
agent
Antitumor
agent
60-90 Å
Shuang, L. Bioconjugate Chem. 2009, 20, 2199-2213
Effective cRGD Conjugate Multimerization
αvβ3 Integrin
60 Å
Xiong, J. P. et al. Science 2002, 296,151-155
PDB1JV2.
Scaffolds in RGD Conjugate Multimerization
Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168 – 2181.
Scaffolds in RGD Conjugate Multimerization
Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168 – 2181.
Multimerization Effect on RGD Conjugate Binding
Competitive binding assay against 125I-echistatin
Immobilized αvβ3
U87MG cells
No. of RGD
Ki [nM]
No. of RGD
Ki [nM]
c(RDG)1
32 ± 6
c(RGD)1
1627 ± 199
c(RGD)2
13 ± 2
c(RGD)2
522 ± 57
c(RGD)4
1.30 ± 0.10
c(RGD)4
248 ± 48
c(RGD)8
0.32 ± 0.04
c(RGD)8
46 ± 5
c(RGD)16
0.26 ± 0.04
c(RGD)16
10 ± 1
U87MG -Human glioblastoma cell line
Carmen, W.; Maschauer, S.; Prante, O. ChemBioChem. 2010, 11, 2168-2181.
RGD Conjugate Design and Effect on Activity
Multimerization
Increase affinity
Immobilization
of RGD Sites
Hydrophilic site
enhance RGDintegrin interaction
Linker Bond
Linker Bond Stability
Amide vs hydrazone
Hydrolyzes slowly
Hydrolyzes fast
Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21, 1779–1787.
Cellular Cleavage of Linker Bonds
Amide bond
Less toxic
Hydrazone bond
More toxic
Christie, R. J.; Anderson, J. D.; Grainger, D. W. Bioconjugate Chem. 2010, 21, 1779–1787.
Amide Linker Bond
Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.
Hydrazone Linker Bond
Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.
Amide vs Hydrazone Bond Linker Stability
Entry
pH 7.4, 37 oC
t1/2, h
pH 5, 37 oC
t1/2, h
1
>72
>72
2a
6
25
2b
5.8
<2 min
Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.
In Vivo Evaluation of Amide Linker Bond Stability
Amide bond linked conjugate in
plasma
Free Drug in plasma of Mice
treated with 1
Amide bond linked conjugate in
tumor
Free Drug in tumor of Mice treated
with 1
Plasma & Tumor drug content
ng/mL plasma & ng/g tissue
100000
10000
1000
100
10
1
0
5
10
15
20
25
30
Time (h)
A2780 -Human ovarian cancer cell line
Amide bond linked conjugates
- Less cytotoxic
Pozzo, A. D. et al. Bioorg & Med. Chem. 2010, 18, 64–72.
Two Linker Bonds in RGD Conjugate System
Drug
RGD
Bond cleavage
Drug
RGD
Bond cleavage
Free drug
Drug derivative
X
Bond stability at pH ~ 5
Drug-Y < Y-X
X-Z < Z-Drug
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Two Linker Bonds in RGD Conjugate System
X – Ester
Y – Hydrazone
Z - Amide
Application in multidrug resistance (MDR)
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Two Linker Bonds in Redirected Drug Delivery
Targets cell nucleus - Prone to Phospho-glycoprotein efflux
Salerno, M.; Przewloka, T.; Fokt, I.; Priebe, W.; Garnier-Suillerot, A. Biochem. Pharmacol. 2002,
63, 1471-1479.
Two Linker Bonds in Redirected Drug Delivery
Targets Mitochondria -No Interaction with Phospho-glycoprotein (p-gp)
Salerno, M.; Przewloka, T.; Fokt, I.; Priebe, W.; Garnier-Suillerot, A. Biochem. Pharmacol. 2002,
63, 1471-1479.
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
In Vitro Assay -Linker Bond Effect on MDR
IC50 Values
Construct
Sensitive
cells
Multi-drug
resistant cells
Free DOX
0.84 g/mL
11.60 μg/mL
Acetal-PEO-b-P(CL-Hyd-DOX)
1.36 μg/mL
-
RGD4C-PEO-b-P(CL-Hyd-DOX)
0.14 μg/mL
7.92 μg/mL
Acetal-PEO-b-P(CL-Ami-DOX)
RGD4C-PEO-b-P(CL-Ami-DOX)
0.73 g/mL
7.24 μg/mL
0.092 μg/mL
MDA-435 /LCC6WT & MDA-435 /LCC6MDR (Breast tumor cell line)
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
In Vivo Studies: Targeted Doxorubicin
Doxorubicin sensitive breast tumor cell line
Mice Survival %
100
80
60
40
20
0
0
10
20
30
40
50
Days after Tumor inoculation
RGD-PEO-b-P(CL-Hyd-DOX)
Control
Free DOX
Acetal- PEO-b-P(CL-Hyd-DOX)
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
In Vivo Studies: Targeted Doxorubicin
Multidrug resistant breast tumor cell line
Mice Survival %
100
80
60
40
20
0
0
10
20
30
40
Days after tumor innoculation
RGD-PEO-b-P(CL-Ami-DOX)
Control
Free DOX
Acetal- PEO-b-P(CL-Ami-DOX)
Xiong, X. B.; Maa, Z.; Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Targeted DOX in Breast Tumor Cell Line
Sensitive cells
Multidrug resistant cells
100
80
Mice Survival %
Mice Survival %
100
60
40
20
0
0
10
20
30
40
Days after Tumor inoculation
RGD-PEO-b-P(CL-Hyd-DOX)
Control
Free DOX
Acetal- PEO-b-P(CL-Hyd-DOX)
50
80
60
40
20
0
0
10
20
30
40
Days after tumor innoculation
RGD-PEO-b-P(CL-Ami-DOX)
Control
Free DOX
Acetal- PEO-b-P(CL-Ami-DOX)
Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Model of Redirected Drug Delivery
Doxorubicin sensitive cells
Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Model of Redirected Drug Delivery
Multidrug resistant cells
Drug efflux
p-gp
Xiong, X. B.; Maa, Z. Lai, R.; Lavasanifar, A. Biomaterials 2010, 31, 757–768.
Outline
 RGD definition
 RGD-Integrin receptors interaction
 Cilengitide®
 RGD conjugate design and effect on activity
 Summary
Checklist
Future directions
Checklist
Criteria
Specificity
Less-Toxic
Low dosage
Non-Targeted Drug cRGD cRGDdelivery
Conjugates
Summary
RGD Tumor Targeting is dependent on the design of the
Conjugate;
 Immobilization site-Binding enhanced on hydrophilic
sites
 Multimerization increase binding affinity
 The linker bond stability - key to reduced toxicity
Future Directions
 Mechanistic studies to explain RGD-conjugate mediated
antitumor agent redirection in MDR and effect of amide linked
RGD conjugate on the functional properties of mitochondria.
 Designing new RGD conjugates that are more soluble by
incorporating a sugar moiety.
• Dr. Walker, Dr. Maleczka, Dr. Huang, Dr. Borhan
• Lab membersChelsea, Danielle, Dilini, Getrude, Irosha, Mark,
Udayanga.
• YCC Panel
• Friends-Camille, David, Philip, Salinda, Washington