1. 1 BTM Nanocapsules for Formulation of Drugs and Vaccines and Imaging Agents Carolina Seeds of Innovation November 4, 2010 Russell J. Mumper, Ph.D. Center for Nanotechnology in Drug Delivery Division of Molecular Pharmaceutics UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill, North Carolina

2. 2 Nanotemplate Engineering Focus Areas Materials (Handbook of Pharmaceutical Excipients) Engineering & Characterization Cell Interaction / Uptake Biofate & Biometabolism Bio- and Hemocompatibility (toxicological aspects of NPs) Cell and Tissue Targeting (therapeutics ) Therapeutic (and Imaging) Areas –Addressing resistance in human cancer using nanotechnology –Subunit (protein) vaccines for HIV

3. 3 Oil Drug (Rx) Surfactant(s) Oil Surfactant(s)Water Clear, Stable Oil-in-Water Microemulsion “Nanotemplate” at 50-65 o C + Water Cool to 25 o C Enables manufacturing of stable NPs <200 nm using a reproducible and scalable process Manufacturing process is as few as 3-steps and is completed in one vessel Overcomes the limitations of commonly used methods to make sub-micron sized particles Step 3Step 2 Step 1 Add, Heat & Mix at 50-65 o C Nanoparticles or Nanocapsules Nanotemplate Engineering oil droplets + _ Ni OH PEG -SH -NH 2 -COOH RXN Y Ligand HO Rx PEG Rx oil droplet nanotemplate = surfactant

4. 4 Vitamin E TPGS (d-Alpha Tocopheryl Polyethylene Glycol 1000 Succinate) CH 3 (CH 2 ) 17 (OCH 2 CH 2 ) 20 OH Brij 78 (Polyoxyethylene 20 stearyl ether) E78 Nanoparticles vs. BTM Nanocapsules Cetyl Alcohol (m.p. 49 o C) CH 3 (CH 2 ) 14 CH 2 OH Miglyol ® 812 Caprylic/Capric Triglyceride C 8 (50-65%); C 10 (30-45%) Oil Phase Surfactants + Solid Lipid E78 NanoparticlesOil-Filled BTM Nanocapsules E78 NPs BTM NPs

5. 5 A New Nanocapsule Formulation - “BTM NPs” First Generation BTM NPs identified by Sequential Simplex Optimization Composition: liquid tri-glyceride core with two surfactants Scalable, one vessel manufacturing process Lipid/drug ~ 20-30:1 w/w; [drug] up to 1.5 mg/ml Sustained-release of drug Can be pegylated to make ‘pegBTM NPs’ Easily sterile filtered Can be lyophilized with no cryoprotectant Very stable in suspension or lyophilized ‘Plug & Play’ platform based on oil properties Very well tolerated, repeated i.v. injection up to 750 mg/kg Dong et al. Eur. J. Pharm. Biopharm. (2009) Lyophilized BTM NPs 15 s after rehydration

6. 6 Mice (n=6/group) were dosed i.v. with PX (4.5 mg/kg) on day 0, 7, 14, and 21 0 200 400 600 800 1000 1200 051015202530 Day Tumor size (mm 3 ) * * ** * * * # Saline Empty BTM NPs (4.5 mg/kg) Taxol (4.5 mg/kg) Taxol (20 mg/kg) Empty BTM NPs + Taxol (4.5 mg/kg) PX BTM NPs (4.5 mg/kg) In-Vivo Efficacy Study in Nude Mice Bearing P-gp+ Resistant Human Ovarian Tumors

7. 7 Retreatment of Mice 0 100 200 300 400 500 600 700 800 900 1000 036912151821242730 0 200 400 600 800 1000 01234567891011 542 303 519 364 340 509 559 420 383 ** * * * * Taxol-failed mice can be salvaged with PX BTM NP treatment Previously PX BTM NP treated mice respond to new course of PX BTM NP treatment Day 4.5 mg/kg 7.5 mg/kg PX BTM dose Tumor size (mm 3 ) Day 31 of Study #2 Day 49 of Study #2

8. 8 Enhancement of Tumor MRI Image with BTM-DTPA-Gd NPs 5 hr after i.v. injection in A549 s.c. xenograft tumors ~50-70 mm 3 Compliments of Dr. Michael Jay in collaboration with SAICF at UNC-BRIC using 9.4T Micro-MRI Control BTM-DTPA-Gd NPs

9. 9 J. Biomed. Nanotech. (2007) Pharmaceutical Research (2007) Vaccine (2004, 2006) HIV/AIDS (2009) Nano-based Subunit HIV Vaccines Dendritic Cell Toll-like Receptor (TLR-9) Receptor MHC I MHC II DC targeting Ligand PEG Tat (1-72) Gag p24 Adjuvant (PRL) 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) Tat & Gag antigens: conserved; critical; CTLs detected in LTNPs NIH-NIAID R01 AI058842 NP benefits: Increased DC uptake/processing/ presentation Dose sparing Enhance MHC1 processing Enhance Th1-type responses Enhance (neutralizing) antibodies Co-delivery of antigen/adjuvant DiOC18 NPs in DC

10. 10 Alum (1 µg) Naive 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 P41 specific IgG O.D. at 450 nm 1,000 2,000 5,000 10,000 E78-Ni (1 µg) BTM-Ni (1 µg) BALB/c mice (n=8/group) were dosed by s.c. injection on day 0 and 14; ELISA day 28 Doses: 0.1, 0.5, or 1  g p41 His-tag p41 provided by Dr. Robert Seder, NIH-NIAID Vaccine Research Center p41 Immunization; BTM-Ni vs. E78-Ni NPs BTM-Ni (0.5 µg) * BTM-Ni (0.1 µg) # * * * # #

11. 11 Concluding Remarks “Nanoparticle Compositions Comprising Liquid Oil Cores” (PCT /US2009/060593 ) “Translational Nanotechnology” – all required elements Nanotemplate Engineering simple, one-vessel process, reproducible, scalable, cost-effective keys: 1) physical chemistry/pharmacy 2) excipient selection Some ‘GRAS’ or USP/NF excipients may be ‘biological modifiers’ Nano-based Drug Delivery Systems Improve drug solubility / bioavailability Address MDR in cancer to improve outcomes Can be used for imaging Co-delivery of antigens / adjuvants for improved vaccines

12. 12 Russell J. Mumper, Ph.D. Center for Nanotechnology in Drug Delivery Division of Molecular Pharmaceutics UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill, North Carolina E-mail: mumper@email.unc.edumumper@email.unc.edu