Nebraska Informatics Center for the Life Sciences
MINI TRACt Session on Bioinformatics
April 20, 2005 Omaha, NE
Alexander Kabanov

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.

 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

Retrovirus Replication
(http://www.accessexcellence.org/AB/GG/retrovirus.html)

Architecture
Linear
Randomly
Branched
Polycation
Linear PEI
(Exgene 500)
Polyethyleneimine
Dendrimer
Graft/block copolymers
Dispersed
Networks
Polyamidoamine
(Superfect TM )
PEOb -PLL
P123g -PEI(2K)
Peptide-PEOg -PEI
PEOcross -PEI
Structure
H
3
C-CH
2
-NH-[CH
2
-CH
2
-NH] n
-CH
2
-CH
2
-NH
3
N
N
NH
PEI
N
N
N
NH
2
N
NH
2
R
O
N
H
N
O
OH
O
NH NH
2
O
N
PEI
O
NH NH
2
PPO
PEO
Pluronic P123
PEG
++
++
++
++
+
PEI
++
+
160 nm
PEO
Peptide

PPO
PEI 2 kDa
PEO
Pluronic P123
DNA
10000
1000
100
10
1
160 nm pCMV-luc, Cos-7 cells, optimized N/P ratio

Pluronic Block
Copolymers
HO CH
2
CH
2
O
EO n/2
CH
2
CHO
CH
3
PO m
CH
2
CH
2
O H n/2
EO
Pluronic F38 EO
40
PO
16
EO
40 m + n = 96
Pluronic P85 EO
26
PO
40
EO
26 m + n = 92
Pluronic F123 EO
19
PO
69
EO
19 m + n = 107
Hydrophobicity increases

HO CH
2
CH
2
O n/2
CH
2
CHO CH
2
CH
2
O H n/2
CH
3 m
SP1017:
Pluronic L61 and Pluronic F127 (1:8) wt.
Naked DNA DNA/SP1017 c57Bl/6
700000
600000
500000
400000
300000
200000
100000
0
*
CMC
0 0.001 0.01
0.05
0.1
1
SP1017 (% w/v)
Lemieux et al. (2000) Gene Therapy 8, 92

2400
2000
1600
1200
800
400
0
0
DNA alone
DNA + P85
10 20
Days
30 40
1800 x 18
DNA alone
DNA + P85 x 11
1200
600
0
5
 g 10
 g x 4
50
 g

1500
1200
900
600
300
0
Balb/C naked DNA
DNA+P85(0.3%)
C57Bl/6 Athymic

Enhanced Gene Expression in Stably Transfected Cells
10000
7500
(-) Pluronic
(+) Pluronic
10
*
5000
2500
3
*
Mouse fibroblasts NIH3T3 were stably transfected with luciferase gene by co-transfection with 5:1 ratio of gWIZluc and phCMV1
0
P85 L64

Luc CMV -NIH3T3 Cells
Control L64 P85
Luc(230bp)
GAPDH (474bp) hsp68 (664bp)
4
3
2
1
0
9
8
7
6
5
HSP/GAPDH
Luc/GAPDH
* *
* *
Media P85
* *
* *
L64

Bacterial,
Viral infection
Stress Cytokines cytoplasm
IκB kinase P-Ikb
B-actin
0 2 5 min
IκB
NFκB
NF-
ATP
Active
κB
NFκB
ADP
I-
κB
P
NFκB
Ubiquitination and degradation of IκB by proteosomes
NFκB
Activation of genes
Nucleus

 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 incorporation into delivery “system”
+
Drug Polymer Delivery “system”
Drug release at the target site
Administer into body
+ Therapeutic effect

Pluronic Block
Copolymers
HO CH
2
CH
2
O
EO n/2
CH
2
CHO
CH
3
PO m
CH
2
CH
2
O H n/2
EO
Pluronic F38 EO
40
PO
16
EO
40 m + n = 96
Pluronic P85 EO
26
PO
40
EO
26 m + n = 92
Pluronic F123 EO
19
PO
69
EO
19 m + n = 107
Hydrophobicity increases

Solubilization
Drug hydrophilic hydrophobic
Micellization
Micelle
Block copolymer
A.V. Kabanov et. al FEBS Lett. 1989, 258, 343-345

MDR in Cancers Tumors
Drug
Pgp
MRP1
Nucleus
GSH/GST
Acidic vesicles
Bcl-2
 p53

Apoptosis

Inhibition of Pgp Efflux
System
0.25
0.2
0.15
0.1
0.05
0
Non resistant
LLC-PK1
Resistant
LLC-MDR1
LLC-PK1 LLC-
MDR1
Assay buffer
0.1% P85
Cells were exposed to
0.1 % P85 for 60 min.
Batrakova et al. (2001) JPET 296, 551

Pluronic Pgp
MRPs
BCRP
Drug
Mitochondria
ATP

GSH/GST
Acidic vesicles
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
100
80
Dnr/P85
SKVLB
Dnr
Dnr/P85
SKOV3
60
40 free Dnr
SKVLB
20
0
0.001
0.1
10
[Daunorubicin], mg/ml
Alakhov et al. (1996) Bioconjugate Chem. 7 , 209

Clinical Trials of Pluronic-
Doxorubicin (SP1049C)
• Phase I completed
• 26 patients in Christie Hospital, Manchester, UK
• MTD 70 mg/m 2
• 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
Technology Company

Prevention of MDR in
MCF7 Breast Carcinoma
Western blot
10000
Dox alone selects resistant cells
1000
100
Dox + P85 no resistance develops
10
1
0 50 100 150 200 250 300
Days
Stepwise increase of the drug concentration: n + 2n
Pgp

-actin

MCF7 MCF7/Dox MCF7/-Dox-
P85
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 (cy3)
MCF7/Dox vs. MCF7:
Upregulated 642 genes
Downregulated: 252 genes
MCF7/Dox-P85 vs. MCF7:
Upregulated 422 genes
Downregulated: 103 genes
MCF7 (cy3)
MCF7/P85 vs. MCF7:
Upregulated 94 genes
Downregulated: 22 genes
UNMC-Eppley Microarray Core Facility (Dr. D. Kelly)

UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)

Multivariate Scatter Plot
UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)

Estrogen dependence
Respiration
Metallothioneins
Heat shock
MDR
Breast cancer resistance
MDR1 transcrition activation
Connective tissue growth
Metabolic resistance


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

• 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

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 National Cancer Institute o National Science Foundation o Nebraska Research Initiative

 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

 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