Transcript Slide 1

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.
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
Outline
 FDA and Nanotechnology
 Nanotemplate Engineering
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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 
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”
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
PharmaceuticallyAcceptable
Matrix @ 40-65C
Clear, Stable
Oil-in-Water (O/W)
Microemulsion
“Nanotemplate”
Let Cool
**
• 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)
Well-Defined,
Uniform, Solid
Nanoparticles
<100 nm
Feasibility Demonstrated
 Engineering & Characterization
 NPs contain up to 80% w/w low Mw drug
 Cell uptake/interaction
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 Increase apparent drug solubility up to 10 -fold  Biometabolism
 Bio- and hemocompatibility
 Coating or entrapment of many types of drugs,  Cell and tissue targeting
antigens, or sensors
 Sterile filtered and pyrogen free; lyophilized
 Hemocompatible to neutrophils, platelets, and RBCs
 Lack of toxicity
 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.
Microemulsions as Precursors to
Solid Nanoparticles
 US Patent # 7,153,525 (12/26/2006)
 Theoretical advantages as an
engineering process:
Process has been scaled to 10 L
100
NPs = 6.7% w/v
90
Oil
80
Particle Size (nm)
 Microemulsions form spontaneously
within the microemulsion ‘window’
 Ideally, add components, heat to form
microemulsion, and then cool
 No special equipment required
 Scalable and reproducible
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60
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40
30
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10
0
5 mL
microemulsion
‘window’
Surfactant(s)
Water
50 mL
0.5 L
glass vessel
using magnetic stirring
1L
5L
10 L
stainless steel vessel with
conventional mechanical mixer
Discovery:
Concentrated NPs can be engineered by
just reducing the volume of water added
Nanotoxicology
 Metabolism: Fatty alcohol-based NPs are
metabolized in-vitro by ADH/NAD+1
 Hemocompatibility of PEG NPs and NPs2
Rationale:
mechanisms & standard methodologies
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2
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No RBC lysis at up to 1 mg/mL
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No neutrophil activation
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No platelet activation/aggregation as
measured with PAC-1 binding
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However, NPs inhibit agonist-induced
platelet activation in a dose-dependent
manner (THR, ADP, IBOP)
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2-3-fold increase in WBCT at >500 mg/mL
Dong & Mumper, Drug Dev. Ind. Pharm. 2006
Koziara et al., Pharm. Res. 2005
Paper profiled by NNI and NCI NCL as an
example of ‘safe nanotechnology’ (Nov. 2005)
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
DAPI = blue
Doxorubicin = red
Fluorescein NPs = green
2 hr influx + 4 hr efflux studies
[DOX] (ng)/[protein] (ug)
Doxorubicin NPs in
Human Breast Cancer Cells
0.6
0.5
NPs >6-fold
increase
in retention
0.4
0.3
0.2
0.1
0
DOX NPs
free DOX
Paclitaxel (PX) NPs Overcome MDR In-Vitro
Human glioblastoma (U-118)
paclitaxel sensitive
Human colon adenocarcinoma (HCT-15)
paclitaxel resistant
IC50 (nM)
viability (%)
75
50
25
150
Taxol 48 h
Taxol 72 h
viability (%)
PX NPs 48 h 22.1 + 9.8
PX NPs 72 h 8.7 + 0.4
Taxol 48 h
21.1 + 8.0
Taxol 72 h
11.5 + 4.1
100
100
PX NPs 72 h
50
PX NPs 48 h
0
0
1
2
log PX conc.
Koziara et al., J. Controlled Rel., 2004
3
0
0
1
2
log PX conc.
3
4
PX NPs Reduce Tumor Growth in Resistant
Mouse Xenograft Model (HCT-15)
tumor volume (mm3)
3500
2000
PX NPs
Taxol
saline
E78 NPs
1500
I.T. dose 1.5 mg/kg
3000
2500
*
1000
*
500
0
3
6
9
12
Time (days)
Koziara et al., J. Controlled Rel., 2006
*
*
0
*#
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Doxorubicin (Dox) NPs Extend Life
in Mouse Syngeneic Resistant Leukemia
Blank NPs
Free Dox
100
% Survival
Control
75
Dox NPs #2
median survival
50
11 12
14.5
16.5
Dox NPs #1
20
25
0
0
3.5 mg Dox/kg
5
10
15
Time (days)
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
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Nanoengineered Vaccines
NPs
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Engineer artificial/synthetic viruses
Safe
Less toxic than other adjuvants
NP
Cost-effective
Enhance solubility/delivery of
vaccine components
Dendritic Cell
Co-delivery of antigen/adjuvant
(APC)
Improve cell uptake and trafficking
Th2
MHC II
NP surface can be functionalized MHC I
CD4+
Th1
Cell-targeting
T cell
CD8+
T cell
IL-2, IL-3,
Increased and/or balanced
IFNg, TNFa
humoral and cellular immune
responses
Cellular
Responses
IFNg
IgG2a
B cell
IL-4
IL-4, IL-5
IL-6, IL-10
Humoral
Responses
IgG1, IgM,
IgA, IgE
Uptake of NPs by DCs
NP uptake in murine BMDDCs
DiOC18 NPs in DC
80
*
70
% uptake
60
*
50
*
40
CTAB NPs (+)
30
SDS NPs (-)
20
Brij 78 NPs (neutral)
10
0
0
2
4
6
8
10
12
14
Time (hr)
*p<0.05
BMDDCs: 2x105/well incubated with 1 mg NPs at 37oC
Study performed at NIH/NIAID/VRC
HIV Vaccine Concept
NIH-NIAID R01 AI058842
Dendritic Cell
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)
Toll-like Receptor
(TLR-9)
MHC I
DC Mannose
Receptor
Tat (1-72)
PEG
Adjuvant
(PRL)
Mannopentaose
(DC targeting)
Gag p24
Tat & Gag p24 antigens: conserved; critical; CTLs detected in LTNPs
Dose Response Study with Tat
Humoral Responses
Total IgG
Cellular Responses
IgG1 = Bold
1000000
150,000
Serum total IgG titer
Serum total IgG titer
175,000
125,000
100,000
75,000
50,000
*
25,000
0.62
0.63
IgG2a / IgG1 ratio
IgG2a = Dashed
0.87
0.22
0.16
100000
*
10000
1000
100
10
0
NPs
5 mg
NPs
1 mg
Alum
5 mg
Alum
1 mg
CFA
5 mg
NPs
5 mg
NPs
1 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
Alum
5 mg
Alum
1 mg
CFA
5 mg
ELISA Pattern of the Anti-Tat Sera
N-terminal
Core Region
LTR transactivation
Cysteine (7) Rich Region
Basic Domain
TAR RNA; nuclear local.
RPPQ
2
20
22
37 38
48 49
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.
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Therapeutic HIV Vaccine Concept
Cell
Immunization
Transactivation
HIV
Y
Abneutralizing
Y Y
cDNA
Tat
1
Antibodies to Tat
Neutralize Tat protein
Maintain low
viral bioburden
Reduce
HIV
transactivation
2
Summary
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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
May, 2009
4th Annual Chapel Hill Drug Conference
“The Use of Nanotechnology to
Create Safe and Effective
Therapeutic and Diagnostic Products”
May, 2009