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Polymer Genomics Alexander Kabanov

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

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Polymer Genomics Alexander Kabanov

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

  2. 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.

  3. Outline • Effects of Synthetic Polymers on Gene Expression during Gene Delivery • Effects of Synthetic Polymers on Gene Expression duringDrug Delivery

  4. Effects of Synthetic Polymers on Gene Expression during Gene Delivery

  5. Retrovirus Replication(http://www.accessexcellence.org/AB/GG/retrovirus.html) Non-Viral Gene Delivery Paradigm

  6. NH NH 2 N N NH 2 N N N N H H N N N H O O OH R O N N NH NH 2 H N O NH NH 2 O PEI PEO Peptide Architecture Polycation Structure Linear PEI (Exgene 500) H3C-CH2-NH-[CH2-CH2-NH]n-CH2-CH2-NH3 Linear Randomly Branched Polyethyleneimine Polyamidoamine (SuperfectTM) Dendrimer Graft/block copolymers PEI PEO-b-PLL P123-g-PEI(2K) Peptide-PEO-g-PEI PEO PPO Pluronic P123 + PEI + PEG Dispersed Networks + + PEO-cross-PEI + + + + + + + 160 nm +

  7. PEI 2 kDa DNA Pluronic P123 PPO 160 nm pCMV-luc, Cos-7 cells, optimized N/P ratio DNA/Polycation Complex PEO

  8. Pluronic Block Copolymers EO PO EO Pluronic F38 EO40-PO16-EO40m + n = 96 Pluronic P85 EO26-PO40-EO26m + n = 92 Pluronic F123 EO19-PO69-EO19m + n = 107 Hydrophobicity increases

  9. Pluronic-Enhanced Gene Expression in Muscle c57Bl/6 SP1017: Pluronic L61 and Pluronic F127 (1:8) wt. Naked DNA DNA/SP1017 CMC Lemieux et al. (2000) Gene Therapy8, 92

  10. 2400 DNA alone DNA + P85 2000 1600 Luciferase/muscle, pg/mg 1200 800 400 0 0 10 20 30 40 Days Enhanced Gene Expression in Muscle DNA alone x 4 DNA + P85 1800 * * x 18 x 11 1200 Luciferase/muscle, pg/mg * * * * 600 0 5 g 10 g 50 g

  11. Genotype Dependence of Pluronic Effect 1500 naked DNA DNA+P85(0.3%) 1200 900 Luciferase/muscle, pg/mg 600 300 0 Balb/C C57Bl/6 Athymic

  12. Promoter-Selectivity of Pluronic Effect

  13. 10 * 3 * 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

  14. Control L64 P85 Luc(230bp) GAPDH (474bp) hsp68 (664bp) * * * * * * * * Effect Pluronic P85 on mRNA Levels in LucCMV-NIH3T3 Cells

  15. 0 2 5 min P-Ikb B-actin P Bacterial, Viral infection Stress Cytokines cytoplasm I-κB kinase I-κB ATP ADP I-κB NF-κB NF-κB Active NF-κB NF-κB Ubiquitination and degradation of I-κB by proteosomes Activation of genes NF-κB Nucleus

  16. 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).

  17. Effects of Synthetic Polymers on Gene Expression during Drug Delivery

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

  19. Pluronic Block Copolymers EO PO EO Pluronic F38 EO40-PO16-EO40m + n = 96 Pluronic P85 EO26-PO40-EO26m + n = 92 Pluronic F123 EO19-PO69-EO19m + n = 107 Hydrophobicity increases

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

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

  22. LLC-PK1 LLC- MDR1 Inhibition of Pgp Efflux System 0.25 0.2 0.15 R123 uptake (nmol/mg prot) 0.1 Cells were exposed to 0.1 % P85 for 60 min. 0.05 Assay buffer 0.1% P85 0 Resistant Non resistant LLC-PK1 LLC-MDR1 Batrakova et al. (2001) JPET 296, 551

  23. MRPs BCRP Pluronic ATP ATP GSH/GST Acidic vesicles BCL2, BCLXL BAX, P53, APAF1, caspases 3, 9 Apoptosis Drug Pgp Mitochondria Nucleus Kabanov et al. (2002) Adv. Drug Del. Rev., 54, 759.

  24. Sensitization of MDR Tumors by Pluronic P85 Dnr/P85 SKVLB Dnr Dnr/P85 SKOV3 free Dnr SKVLB Alakhov et al. (1996) Bioconjugate Chem.7, 209

  25. 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

  26. Prevention of MDR in MCF7 Breast Carcinoma Western blot Dox alone selects resistant cells Pgp -actin Dox + P85 no resistance develops MCF7 MCF7/200 MCF7/1000 MCF7/10P85 Stepwise increase of the drug concentration: n + 2n

  27. MCF7 MCF7/Dox MCF7/-Dox-P85 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.

  28. Global (20K) Gene Expression MCF7/P85 (cy5) MCF7/P85 (cy5) MCF7/P85 (cy5) MCF7 (cy3) MCF7 (cy3) MCF7 (cy3) MCF7/Dox vs. MCF7: Upregulated 642genes Downregulated:252genes MCF7/Dox-P85 vs. MCF7: Upregulated 422genes Downregulated:103genes MCF7/P85 vs. MCF7: Upregulated 94genes Downregulated:22genes UNMC-Eppley Microarray Core Facility (Dr. D. Kelly)

  29. Self-Organizing Map (SOM) Analysis UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)

  30. Dox vs. Pluronic/Dox Selected Cells Multivariate Scatter Plot UNMC Eppley Bioinformatics Shared Resource Drs. Sherman, Xiao)

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

  32. 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

  33. 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

  34. Acknowledgement • National Cancer Institute • National Science Foundation • Nebraska Research Initiative 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

  35. 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

  36. 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

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