polymer genomics alexander kabanov
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Nebraska Informatics Center for the Life Sciences MINI TRACt Session on Bioinformatics April 20, 2005 Omaha, NE. Polymer Genomics Alexander Kabanov. Drug Delivery. - PowerPoint PPT PresentationTRANSCRIPT
Polymer GenomicsPolymer Genomics
Alexander Kabanov
Nebraska Informatics Center for the Life SciencesMINI TRACt Session on BioinformaticsApril 20, 2005 Omaha, NE
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.
Drug Delivery
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.ht
ml)
Randomly Branched
PEO-b-PLLP123-g-PEI(2K)
Peptide-PEO-g-PEI
Graft/block copolymers
Polyamidoamine (SuperfectTM)Dendrimer
Linear PEI(Exgene 500)
H3C-CH2-NH-[CH2-CH2-NH]n-CH2-CH2-NH3
Polyethyleneimine
StructurePolycationArchitecture
RNH
N
O OHO
NO
ONH NH2
NHO
NH2
Linear
Dispersed Networks
PEO-cross-PEI
Pluronic P123
PEI
PPO
PEO
PEI
PEO
Peptide
160 nm
+++
+++
++++++
PEIPEG
NH
N N N NH2
N
NH2
HN
NN
NH
NH
DNA/Polycation ComplexPEI 2 kDa
PPO
PEO
Pluronic P123
160 nm
1
10
100
1000
10000
ExG
en™
50
0
Sup
erfe
ct™
F1
23
-g-
PE
I(2K
)
PE
I (25
K)
PE
I (50
K)
PE
VP
36
2
PE
O-g
-PE
I
Luc
ifera
se (
ng/m
g)
pCMV-luc, Cos-7 cells, optimized N/P ratio
DNA
Pluronic Block Copolymers
Pluronic F38 EO40-PO16-EO40 m + n = 96
Pluronic P85 EO26-PO40-EO26 m + n = 92
Pluronic F123 EO19-PO69-EO19 m + n = 107
HO CH2CH2O CH2CHO
CH3
CH2CH2O Hn/2 n/2
m
EO EOPO
Hydrophobicityincreases
Pluronic-Enhanced Gene Pluronic-Enhanced Gene Expression in Muscle Expression in Muscle
Lemieux et al. (2000) Gene Therapy 8, 92
HO CH2CH2O CH2CHO
CH3
CH2CH2O Hn/2 n/2
mc57Bl/6
SP1017: Pluronic L61 and Pluronic F127 (1:8) wt.
CMC
Naked DNA DNA/SP1017
Enhanced Gene Enhanced Gene Expression in Muscle Expression in Muscle
DNA alone
DNA + P85
x 11
x 4
0
600
1200
1800
5 g 10 g 50 gLu
cife
rase
/mus
cle
, pg
/mg
x 18
DNA alone
DNA + P85
0
400
800
1200
1600
2000
2400
0 10 20 30 40
Days
Luci
fera
se/m
uscl
e,
pg/m
g
Genotype Dependence of Genotype Dependence of Pluronic Effect Pluronic Effect
0
300
600
900
1200
1500
Balb/C C57Bl/6 Athymic
Luci
fera
se/m
uscl
e,
pg/m
g naked DNADNA+P85(0.3%)
Promoter-Selectivity of Promoter-Selectivity of Pluronic EffectPluronic Effect
Enhanced Gene Expression in Stably Transfected Cells
Mouse fibroblasts NIH3T3 were stably transfected with luciferase gene by co-transfection with 5:1 ratio of gWIZluc and phCMV1
0
2500
5000
7500
10000
P85 L64
Luci
fera
se,
pg/m
g ce
ll pr
otei
n (-) Pluronic
(+) Pluronic
3
10
*
*
Effect Pluronic P85 on Effect Pluronic P85 on mRNA Levels inmRNA Levels in
0
1
2
3
4
5
6
7
8
9
Media P85 L64
arbi
trar
y un
its
HSP/GAPDH
Luc/GAPDH
* *
* *
* *
* * Control L64 P85
Luc(230bp)
GAPDH (474bp)
hsp68 (664bp)
LucLucCMVCMV-NIH3T3 Cells-NIH3T3 Cells
Stress Cytokines
Bacterial,Viral
infection
I-κB kinase
ATP ADPNF-κB
I-κB
NF-κB
I-κBP
NF-κBUbiquitination and degradation of I-κB by proteosomes
Active NF-κB
Nucleus
cytoplasm
Activation of genesNF-κB
P-IkbB-actin
0 2 5 min
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
DrugAdminister into bodyPolymer Delivery “system”
+
Drug incorporation into delivery “system”
Drug release at the target site
+ Therapeutic effect
Pluronic Block Copolymers
Pluronic F38 EO40-PO16-EO40 m + n = 96
Pluronic P85 EO26-PO40-EO26 m + n = 92
Pluronic F123 EO19-PO69-EO19 m + n = 107
HO CH2CH2O CH2CHO
CH3
CH2CH2O Hn/2 n/2
m
EO EOPO
Hydrophobicityincreases
A.V. Kabanov et. al FEBS Lett. 1989, 258, 343-345
hydrophilic
hydrophobic
Micellar Nanocontainers
Micelle
Block copolymer
DrugSolubilization
Micellization
MDR in Cancers Tumors
Acidic vesicles
Nucleus
Pgp
Apoptosis
MRP1Drug
GSH/GST
Bcl-2p53
Inhibition of Pgp Efflux System
Cells were exposed to 0.1 % P85 for 60 min.
Batrakova et al. (2001) JPET 296, 551
0
0.05
0.1
0.15
0.2
0.25
Non resistantLLC-PK1
ResistantLLC-MDR1
R12
3 up
take
(nm
ol/m
g pr
ot)
LLC-PK1 LLC-MDR1
Assay buffer
0.1% P85
Nucleus
PgpDrugDrug
Mitochondria
ATPATP
PluronicPluronic
Kabanov et al. (2002) Adv. Drug Del. Rev., 54, 759.
Acidic vesicles
MRPsBCRP
GSH/GST
Apoptosis
BCL2, BCLXL
BAX, P53, APAF1,caspases 3, 9
Sensitization of MDR Tumors by Pluronic P85
Alakhov et al. (1996) Bioconjugate Chem. 7, 209
0
20
40
60
80
100
0.001 0.1 10
[Daunorubicin], mg/ml
Inhi
bitio
n, %
0
20
40
60
80
100
0.001 0.1 10
[Daunorubicin], mg/ml
Inhi
bitio
n, %
free DnrSKVLB
Dnr/P85SKVLB
DnrDnr/P85SKOV3
Clinical Trials of Pluronic-Doxorubicin (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
The BiotransportThe BiotransportThe BiotransportTMTMTM Technology Company Technology Company Technology Company
Prevention of MDR in MCF7 Breast Carcinoma
Pgp
-actin
MCF7
MCF7/10P85
MCF7/200
MCF7/1000
Western blot
Stepwise increase of the drug concentration: n + 2n
Dox alone selects resistant cells
Dox + P85 no resistance develops
Selected and Parental Breast Cancer Cells
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/Dox
MCF7 MCF7/-Dox-P85
MCF7/Dox vs. MCF7: Upregulated 642 genesDownregulated: 252 genes
MCF7/P85 vs. MCF7: Upregulated 94 genesDownregulated: 22 genes
MCF7/Dox-P85 vs. MCF7: Upregulated 422 genesDownregulated: 103 genes
UNMC-Eppley Microarray Core Facility (Dr. D. Kelly)
Global (20K) Gene Expression
MC
F7/
P85
(cy
5)
MC
F7/
P85
(cy
5)
MC
F7/
P85
(cy
5)
MCF7 (cy3) MCF7 (cy3) MCF7 (cy3)
Self-Organizing Map (SOM) Analysis
UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)
Dox vs. Pluronic/Dox Selected Cells
UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)
Multivariate Scatter Plot
MDR
Breast cancer resistance
MDR1 transcrition activation
Connective tissue growth
Metabolic resistanceEstrogen
dependence
Metallothioneins
Heat shock
Respiration
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 BatrakovaZhihui YanJian ZhuSrikanth SriadibhatlaShu LiCatherine GebhartDavid KelleySimon ShermanLi Xiao
Supratek Pharma Inc.:Valery Alakhov
o National Cancer Instituteo National Science Foundationo Nebraska Research Initiative
Moscow state UniversityDasha Alakhova
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