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