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Russell J. Mumper, Ph.D.

Russell J. Mumper, Ph.D. Director: Center for Nanotechnology in Drug Delivery at the University of North Carolina - Chapel Hill. Moving Toward Clinical Investigation with Pharmaceutically Engineered Lipid-Based Nanoparticles. Russell J. Mumper, Ph.D.

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Russell J. Mumper, Ph.D.

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  1. Russell J. Mumper, Ph.D. Director: Center for Nanotechnology in Drug Delivery at the University of North Carolina - Chapel Hill

  2. Moving Toward Clinical Investigation with Pharmaceutically Engineered Lipid-Based Nanoparticles Russell J. Mumper, Ph.D. Center for Nanotechnology in Drug Delivery Division of Molecular Pharmaceutics School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill, North Carolina 1st Annual Unither Nanomedical & Telemedical Technology Conference April 1-4, 2008

  3. Outline FDA and Nanotechnology Nanotemplate Engineering NP engineering & characterization NP cell uptake/interaction NP biometabolism NP bio- and hemocompatibility NP cell and tissue targeting Cancer Therapeutics Addressing multi-drug resistance Vaccine Delivery Systems Dendritic cell targeting Nanoengineered subunit HIV vaccine • Cell and Tissue Targeting • Resistant Cancer  • Dendritic Cells 

  4. The FDA and Nanotechnology The FDA has not established its own definition of ‘nano’ Using the NNI’s definition FDA’s Nanotechnology Report dated July 25, 2007 (38 pg.) Initial recommendation: ‘focus on improving scientific knowledge’ • FDA is currently referring to the 2002 Guidance Document on “Liposome Drug Products” • Liposomes are “intended to exhibit a different pharmacokinetic and/or tissue distribution (PK/TD) profile”

  5. Nanotemplate Engineering • Enables manufacturing of stable nanoparticles <100 nm using a one-step, reproducible, and scalable process • Manufacturing process that overcomes the limitations of commonly-used methods to make sub-micron sized particles Add Surfactant** Melt Pharmaceutically- Acceptable Matrix @ 40-65C Clear, Stable Oil-in-Water (O/W) Microemulsion “Nanotemplate” Let Cool Well-Defined, Uniform, Solid Nanoparticles <100 nm • ** • Cationic or anionic surfactants (+/- NPs) • Pegylated surfactants (PEG-NPs) • Amine-reactive surfactants (Amine-NPs) • Sulfhydryl-reactive surfactants (SH-NPs) • Nickel-chelated surfactants (Ni-NPs) • Ligand-derivatized surfactant (Ligand-NPs)

  6. Feasibility Demonstrated NPs contain up to 80% w/w low Mw drug Increase apparent drug solubility up to 105-fold Coating or entrapment of many types of drugs, antigens, or sensors Sterile filtered and pyrogen free; lyophilized Hemocompatible to neutrophils, platelets, and RBCs Lack of toxicity • Engineering & Characterization • Cell uptake/interaction • Biometabolism • Bio- and hemocompatibility • Cell and tissue targeting • Enhanced oral delivery (Dr. Michael Jay; UK) • MRI imaging agent (Dr. Michael Jay; UK) • Transport across blood-brain barrier (BBB) • In-vivo (genetic) vaccines – enhanced responses by multiple routes • In-vivo targeting to solid tumors (overcome MDR) Past 6 years: ~$5 million in funding >40 scientific papers NanoMed Pharmaceuticals, Inc.

  7. Oil 100 microemulsion ‘window’ 90 80 70 60 50 Particle Size (nm) 40 Water Surfactant(s) 30 20 10 0 5 mL 50 mL 0.5 L 1 L 5 L 10 L glass vessel using magnetic stirring stainless steel vessel with conventional mechanical mixer Microemulsions as Precursors to Solid Nanoparticles • US Patent # 7,153,525 (12/26/2006) • Theoretical advantages as an engineering process: • Microemulsions form spontaneously within the microemulsion ‘window’ • Ideally, add components, heat to form microemulsion, and then cool • No special equipment required • Scalable and reproducible Process has been scaled to 10 L NPs = 6.7% w/v Discovery: Concentrated NPs can be engineered by just reducing the volume of water added

  8. Nanotoxicology • Metabolism: Fatty alcohol-based NPs are metabolized in-vitro by ADH/NAD+1 • Hemocompatibility of PEG NPs and NPs2 • Rationale: • mechanisms&standard methodologies • No RBC lysis at up to 1 mg/mL • No neutrophil activation • No platelet activation/aggregation as measured with PAC-1 binding • However, NPs inhibit agonist-induced platelet activation in a dose-dependent manner (THR, ADP, IBOP) • 2-3-fold increase in WBCT at >500 mg/mL Paper profiled by NNI and NCI NCL as an example of ‘safe nanotechnology’ (Nov. 2005) 1 Dong & Mumper, Drug Dev. Ind. Pharm. 2006 2 Koziara et al., Pharm. Res. 2005

  9. DAPI = blue Doxorubicin = red Fluorescein NPs = green NIH-NCI R01 CA115197 Using Nanotechnology to Target Cancer • NPs significantly reduce IC50 (2-10 fold) in several different resistant cell lines • Paclitaxel, doxorubicin, idarubicin, others • Leukemia, breast, ovarian, colon, melanoma • In-vitro results have been translated to xenograft and syngeneic mouse models • Mechanisms: increased uptake/retention, p-gp inhibition, ATP depletion Doxorubicin NPs in Human Breast Cancer Cells 2 hr influx + 4 hr efflux studies NPs >6-fold increase in retention

  10. Paclitaxel (PX) NPs Overcome MDR In-Vitro IC50 (nM) PX NPs 48 h 22.1 + 9.8 PX NPs 72 h 8.7 + 0.4 150 100 Taxol 48 h Taxol 48 h 21.1 + 8.0 Taxol 72 h 75 100 viability (%) viability (%) 50 PX NPs 72 h 50 25 PX NPs 48 h 0 0 0 1 2 3 0 1 2 3 4 log PX conc. log PX conc. Human glioblastoma (U-118) paclitaxel sensitive Human colon adenocarcinoma (HCT-15) paclitaxel resistant Taxol 72 h 11.5 + 4.1 Koziara et al., J. Controlled Rel., 2004

  11. 3500 PX NPs 3000 Taxol saline 2500 E78 NPs 2000 tumor volume (mm3) I.T. dose 1.5 mg/kg 1500 * 1000 * # * 500 * * 0 0 3 6 9 12 15 18 21 Time (days) PX NPs Reduce Tumor Growth in Resistant Mouse Xenograft Model (HCT-15) Koziara et al., J. Controlled Rel., 2006

  12. Doxorubicin (Dox) NPs Extend Life in Mouse Syngeneic Resistant Leukemia Blank NPs Free Dox Control Dox NPs #2 Dox NPs #1 100 75 16.5 14.5 % Survival 11 12 20 median survival 50 25 0 0 5 10 15 20 25 Time (days) 3.5 mg Dox/kg Doxorubicin resistant P388/Adr leukemia cells CD2F1 mice were implanted i.p. with 1 x 105 P388/Adr cells Control = 10 mice; All other groups = 6 mice/group

  13. NP Nanoengineered Vaccines NPs • Engineer artificial/synthetic viruses • Safe • Less toxic than other adjuvants • Cost-effective • Enhance solubility/delivery of vaccine components • Co-delivery of antigen/adjuvant • Improve cell uptake and trafficking • NP surface can be functionalized • Cell-targeting • Increased and/or balanced humoral and cellular immune responses IgG2a IFNg Dendritic Cell (APC) B cell IgG1, IgM, IgA, IgE IL-4 Th2 MHC II MHC I IL-4, IL-5 IL-6, IL-10 CD4+ T cell Th1 CD8+ T cell IL-2, IL-3, IFNg, TNFa Humoral Responses Cellular Responses

  14. Uptake of NPs by DCs NP uptake in murine BMDDCs DiOC18 NPs in DC 80 * 70 60 * 50 % uptake * 40 CTAB NPs (+) 30 SDS NPs (-) 20 Brij 78 NPs (neutral) 10 0 0 2 4 6 8 10 12 14 Time (hr) Study performed at NIH/NIAID/VRC *p<0.05 BMDDCs: 2x105/well incubated with 1 mg NPs at 37oC

  15. HIV Vaccine Concept A DC-targeted nanoparticle with conserved proteins Tat (1-72) and Gag p24 to generate protective Th1, CTL, and neutralizing antibody responses that may be further enhanced by co-delivery of Adjuvants (PRLs) NIH-NIAID R01 AI058842 Dendritic Cell Toll-like Receptor (TLR-9) MHC I DC Mannose Receptor MHC II Tat (1-72) PEG Adjuvant (PRL) Mannopentaose (DC targeting) Gag p24 Tat & Gag p24 antigens: conserved; critical; CTLs detected in LTNPs

  16. Dose Response Study with Tat Humoral Responses Total IgG Cellular Responses IgG2a / IgG1 ratio IgG1 = Bold IgG2a = Dashed 175,000 0.87 0.62 0.63 0.22 1000000 150,000 0.16 * 100000 125,000 100,000 10000 Serum total IgG titer Serum total IgG titer 75,000 1000 50,000 * 100 25,000 10 0 NPs 5 mg NPs 1 mg Alum 5 mg Alum 1 mg CFA 5 mg NPs 5 mg NPs 1 mg Alum 5 mg Alum 1 mg CFA 5 mg BALB/c mice (n=5-6) immunized SC on day 0, 21, and 28; Sac day 35; * p<0.05 Cui et al., Vaccine. 2004; Patel et al., Vaccine. 2006

  17. ELISA Pattern of the Anti-Tat Sera Core Region LTR transactivation Basic Domain TAR RNA; nuclear local. N-terminal Cysteine (7) Rich Region RPPQ 2 20 22 37 38 48 49 57 61 OD450 (1:100 dilution) Cutoff = average naive response + (3*Std. Dev.); (-) no responses BALB/c mice (n=5-6) immunized SC with 5 mg Tat on day 0, 21, and 28; Sac day 35; Tat peptides: NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

  18. Therapeutic HIV Vaccine Concept Cell Immunization Transactivation HIV cDNA Y Tat 2 Y Y Abneutralizing 1 Y Y Antibodies to Tat Neutralize Tat protein Reduce HIV transactivation Maintain low viral bioburden

  19. Summary • Engineering & Characterization • Cell uptake/interaction • Biometabolism • Bio- and hemocompatibility • Cell and tissue targeting • Highly uniform lipid NPs <100 nm can be engineered using a warm o/w microemulsions as a scalable precursor • Nanoparticles: enhance solubility, stability, uptake and delivery • Nanoparticles are hemocompatible & metabolizable • Vaccines: dose sparing, enhance MHC1 processing, enhance Th1-type responses, enhance (neutralizing) antibodies, co-delivery of antigen/adjuvant • Focus on nanoengineered HIV vaccines (Tat + Gag 24) • Cancer: nanoparticles can overcome MDR in-vitro and in-vivo • Focus on reformulating otherwise effective cytotoxic agents

  20. May, 2009 4th Annual Chapel Hill Drug Conference “The Use of Nanotechnology to Create Safe and Effective Therapeutic and Diagnostic Products” May, 2009

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