Download nicls

Nebraska Informatics Center for the Life Sciences
MINI TRACt Session on Bioinformatics
April 20, 2005 Omaha, NE
Polymer Genomics
Alexander Kabanov
Drug Delivery
Transport of a drug, protein or
DNA to its critical site of action
within the body at concentrations
sufficient to produce the desired
therapeutic effect.
Outline
Effects of Synthetic Polymers on Gene
Expression during Gene Delivery
 Effects of Synthetic Polymers on Gene
Expression during Drug Delivery

Effects of Synthetic Polymers
on Gene Expression during
Gene Delivery
Non-Viral Gene Delivery
Paradigm
Retrovirus Replication
(http://www.accessexcellence.org/AB/GG/retrovirus.html)
Architecture
Linear
Randomly
Branched
Polycation
Structure
Linear PEI
(Exgene 500)
H3C-CH2-NH-[CH2-CH2-NH]n-CH2-CH2-NH3
NH
Polyethyleneimine
N
N
NH2
N
N N
H
N
Dendrimer
Polyamidoamine
(SuperfectTM)
R
H
N
N
H
O
O
N
H
NH2
N
OH
N
O
O
Graft/block
copolymers
PEO-b-PLL
P123-g-PEI(2K)
Peptide-PEO-g-PEI
NH 2
PEO
Peptide
Pluronic P123
PEG
Dispersed
Networks
NH
PEI
PEO
PPO
NH 2
N
O
PEI
NH
PEO-cross-PEI
++
++
++
++ PEI
++
++
160 nm
DNA/Polycation Complex
PEI 2 kDa
DNA
PEO
Pluronic P123
PPO
1000
100
10
Luciferase (ng/mg)
10000
1
ExGen™
500
Superfect™
F123-gPEI(2K)
PEI (25K)
PEI (50K)
PEVP362
PEO-g-PEI
160 nm
pCMV-luc, Cos-7 cells, optimized N/P ratio
Pluronic Block
Copolymers
HO
CH2CH2O
CH2CHO
CH2CH2O
n/2
n/2
CH3
EO
H
PO
m
EO
Pluronic F38
EO40-PO16-EO40
m + n = 96
Pluronic P85
EO26-PO40-EO26
m + n = 92
Pluronic F123
EO19-PO69-EO19
m + n = 107
Hydrophobicity
increases
Pluronic-Enhanced Gene
Expression in Muscle
CH2CH2O
CH2CHO
CH2CH2O
n/2
H
CH3
m
SP1017:
Pluronic L61 and Pluronic F127 (1:8) wt.
Naked DNA
c57Bl/6
n/2
DNA/SP1017
700000
*
600000
RLU/s/t.a. muscle
HO
500000
400000
300000
CMC
200000
100000
0
0
0.001 0.01 0.05
0.1
1
SP1017 (% w/v)
Lemieux et al. (2000) Gene Therapy 8, 92
DNA alone
DNA alone
2400
Luciferase/muscle, pg/mg
Luciferase/muscle, pg/mg
Enhanced Gene
Expression in Muscle
DNA + P85
2000
1600
1200
800
400
0
0
10
20
Days
30
40
x4
DNA + P85
1800
*
x 18
x 11
1200
*
*
600
0
5 g
10 g
50 g
Genotype Dependence of
Pluronic Effect
Luciferase/muscle, pg/mg
1500
naked DNA
DNA+P85(0.3%)
1200
900
600
300
0
Balb/C
C57Bl/6
Athymic
Promoter-Selectivity of
Pluronic Effect
Enhanced Gene Expression
in Stably Transfected Cells
10
10000
Mouse fibroblasts NIH3T3 were
stably transfected with luciferase
gene by co-transfection with 5:1
ratio of gWIZluc and phCMV1
Luciferase, pg/mg cell protein
*
7500
5000
(-) Pluronic
(+) Pluronic
3
*
2500
0
P85
L64
Effect Pluronic P85 on
mRNA Levels in
LucCMV-NIH3T3 Cells
L64
9
P85
8
Luc(230bp)
7
GAPDH (474bp)
hsp68 (664bp)
arbitrary units
Control
6
**
HSP/GAPDH
Luc/GAPDH
**
5
4
**
3
**
2
1
0
Media
P85
L64
Bacterial,
Viral
infection
Stress
Cytokines
0
cytoplasm
I-κB kinase
NF-κB
ADP
5 min
P-Ikb
B-actin
I-κB
ATP
2
I-κB
P
NF-κB
Active NF-κB
NF-κB
Activation of genes
NF-κB
Nucleus
Ubiquitination and
degradation of I-κB
by proteosomes
Conclusion

Pluronic block copolymers can increase expression of genes
that are already present in the cells through mechanism(s)
other than enhanced DNA delivery

The mechanism involves activation of transcription

This effect is promoter selective and involves selected
signaling pathways (NF-kB, p53).
Effects of Synthetic Polymers
on Gene Expression during
Drug Delivery
Drug Delivery Concept
Drug incorporation into delivery “system”
+
Drug
Polymer
Administer
into body
Delivery “system”
Drug release at the target site
+
Therapeutic
effect
Pluronic Block
Copolymers
HO
CH2CH2O
CH2CHO
CH2CH2O
n/2
n/2
CH3
EO
H
PO
m
EO
Pluronic F38
EO40-PO16-EO40
m + n = 96
Pluronic P85
EO26-PO40-EO26
m + n = 92
Pluronic F123
EO19-PO69-EO19
m + n = 107
Hydrophobicity
increases
Micellar Nanocontainers
Solubilization
Drug
Micellization
Micelle
hydrophilic
hydrophobic
Block copolymer
A.V. Kabanov et. al FEBS Lett. 1989, 258, 343-345
MDR in Cancers Tumors
Pgp
Drug
MRP1
GSH/GST
Nucleus
Acidic
vesicles
Bcl-2
p53
Apoptosis
R123 uptake (nmol/mg prot)
Inhibition of Pgp Efflux
System
0.25
0.2
0.15
LLC-PK1 LLCMDR1
0.1
0.05
Assay buffer
0.1% P85
0
Non resistant Resistant
LLC-PK1
LLC-MDR1
Cells were
exposed to
0.1 % P85
for 60 min.
Batrakova et al. (2001) JPET 296, 551
Pgp
Pluronic
ATP
ATP
Drug
MRPs
BCRP
GSH/GST
Acidic
vesicles
Mitochondria
BCL2, BCLXL
BAX, P53, APAF1,
caspases 3, 9
Nucleus
Apoptosis
Kabanov et al. (2002) Adv. Drug Del. Rev., 54, 759.
Sensitization of MDR
Tumors by Pluronic P85
Inhibition, %
100
Dnr/P85
SKVLB
Dnr
Dnr/P85
SKOV3
80
60
free Dnr
SKVLB
40
20
0
0.001
0.1
10
[Daunorubicin], mg/ml
Alakhov et al. (1996) Bioconjugate Chem. 7, 209
Clinical Trials of PluronicDoxorubicin (SP1049C)
• Phase I completed
• 26 patients in Christie Hospital, Manchester, UK
• MTD 70 mg/m2
• Anti-tumor activity in some patients with advanced
solid tumors
(Danson et al. 2004, Br. J. Cancer, 90: 2085)
• Phase II trial in progress
• Inoperable metastatic adenocarcinoma of the
esophagus
TM
The BiotransportTM
Technology Company
Prevention of MDR in
MCF7 Breast Carcinoma
Dox alone selects
resistant cells
[Dox], ng/ml
10000
1000
Western blot
Pgp
-actin
Dox + P85 no
resistance
develops
100
10
1
0
50 100 150 200 250 300
Days
Stepwise increase of the drug
concentration: n + 2n
Selected and Parental
Breast Cancer Cells
MCF7
MCF7/Dox
MCF7/-DoxP85
Human breast carcinoma cells, MCF7 were selected by exposure to increasing
concentrations of Dox (MCF7/1000), or Dox/Pluronic P85 (MCF7/10P85)
Simultaneous visualization of F- and G-actin using F-actin–specific Oregon Green 488
phalloidin and G-actin–specific Texas Red deoxyribonuclease I.
MCF7 (cy3)
MCF7/P85 (cy5)
MCF7/P85 (cy5)
MCF7/P85 (cy5)
Global (20K) Gene
Expression
MCF7 (cy3)
MCF7/Dox vs. MCF7:
MCF7/Dox-P85 vs. MCF7:
Upregulated 642 genes
Upregulated 422 genes
Downregulated: 252 genes Downregulated: 103 genes
MCF7 (cy3)
MCF7/P85 vs. MCF7:
Upregulated 94 genes
Downregulated: 22 genes
UNMC-Eppley Microarray Core Facility (Dr. D. Kelly)
Self-Organizing Map
(SOM) Analysis
UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)
Dox vs. Pluronic/Dox
Selected Cells
Multivariate Scatter Plot
UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)
Respiration
Metallothioneins
Heat shock
MDR
Breast cancer
resistance
MDR1 transcrition
activation
Connective
tissue growth
Estrogen
dependence
Metabolic
resistance
Conclusion

Pluronic block copolymers alone are “genetically
benign”

When combined with a drug they can alter gene
expression during selection of cancer cells

They can prevent development of drug resistance for
example MDR in breast tumors

Some genes are altered with drug/polymer
formulation that are not altered with drug alone
Polymer Genomics
Hypothesis
•
Select polymers that alone are
genetically benign when combined with
“biological agents” (low molecular mass
drugs, antigens, DNA) can alter specific
genomic responses to these agents.
•
•
These polymers should have a “weak phenotypic
effect” on cells, e.g. be membrane-active, such as
water-soluble amphiphilic block copolymers and
polyelectrolytes
These polymers perhaps act by interfering with
cell signaling mechanisms
Acknowledgement
UNMC:
Elena Batrakova
Zhihui Yan
Jian Zhu
Srikanth Sriadibhatla
Shu Li
Catherine Gebhart
David Kelley
Simon Sherman
Li Xiao
Supratek Pharma Inc.:
Valery Alakhov
Moscow state University
Dasha Alakhova
o
o
o
National Cancer Institute
National Science Foundation
Nebraska Research Initiative
Conclusion
 Pluronic displays selective activity
towards cells expressing MDR1 gene
 MDR1 gene expression is a valuable
marker to predict success of
Pluronic/drug formulation in cancer
Conclusion
 Pluronic alters the transcript levels
expressed in cancer cells in response to
chemotherapy and in particular
abolishes development of MDR, which
may be an additional benefit in cancer
therapy
 Certain genes are altered with
drug/Pluronic that are not affected with
the drug alone