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Advances in Nano Drugs for Cancer
Chemotherapy: Biopharmaceutical Trends
and Perspectives and Case Studies
Panayiotis P. Constantinides, Ph.D
Biopharmaceutical & Drug Delivery Consulting, LLC
Gurnee, Illinois, USA
[email protected]
Keynote Forum
3rd International Conference and Exhibition on
Pharmaceutics & Novel Drug Delivery Systems
April 8-10, Northbrook, Illinois
OUTLINE
• Biopharmaceutical Aspects of Anticancer Nano Drugs
–
–
–
–
Image-guided drug delivery and multifunctional nanoparticles
Nanoparticle targeting principles
Marketed drug products and in development
Formulation development, manufacturing, Toxicity/PK/ADME
• Case Studies
• A. Parenteral Nano drugs
– Nanoemulsions : paclitaxel
– Liposomes and polymeric micelles : phospho-Ibuprofen (NME)
• B. Oral Nano drugs
– SNEDDS : Tamoxifen
– Reverse Micelles : Leuprolide
• Conclusions and Future Perspectives
4/8/2013
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BIOPHARMACEUTICAL ASPECTS OF
ANTICANCER NANODRUGS
4/8/2013
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Non-invasively
assess drug site
accumulation
Monitor and quantify
drug release
Visualize
biodistribution in
real time
Image-guided
Drug Delivery
Analyze drug
distribution at
the target site
Predict drug
response
Evaluate drug
efficacy
longitudinally
Facilitate
triggered drug
release
Combine disease
diagnosis and therapy
Lammers, T. et al; Mol. Pharmaceutics 7 (6) : 1899-1912 (2010)
4/8/2013
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Morphology and Function of a Multifunctional Nanoparticle
R. Heskel, P.P. Constantinides and D. Sun, AAPS NewsMagazine, January 2012
D
C
+/–
E
A
F
B
(A)
(B)
(C)
(D)
(E)
(F)
Core: drug, magnetic (MRI), quantum dot (optical imaging)
Shell: drug/gene, lipid, polymer (intracellular targeting)
Polymeric Stabilizer: osmotic, entropic, steric
Biological (“Stealth”) Stabilizer: PEG
Surfactant Stabilizer: electrostatic, osmotic
Targeting Moiety: antibody, aptamer, ligand
4/8/2013
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Nanodimensions of Drug Delivery Nanoparticles
Mattheolabakis, G., Rigas B., and Constantinides, P.P. Nanomedicine (2012) 7: 1577-1590
The true nanorange is narrowly
defined as the 1-100 nm
particles.
Marketed injectable liposomal
(DaunoXome®, Doxil®) and
albumin-bound nanoparticles
(Abraxane®) anticancer drug
products, as well as the oral
NanoCrystal® drug products
(Rapamune®, EMEND®, TriCor®
145, Megace® ES and INVEGA®
SUSTENNA®) are within the
submicron range (100 – 1000 nm).
4/8/2013
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Drug Targeting
Active
Passive
• Liver and spleen
• Receptor- mediated
– Immunotargeting
• Antigenic sites on
pathogens
• Infected cells expressing
antigenic structures
• Tumor-associated
antigens
– Targeting Ligands
• EGF, Transferrin
• Folate, RGD
• VIP
4/8/2013
– Macrophage uptake
• Lysosomal enzyme
deficiencies
• Size-mediated
– EPR effect
• pH-mediated
– pH-responsive drug carriers
• Temperature-mediated
– Thermo-responsive drug
carriers
BPDDC LLC www.bpddc.com
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Diverse Targeted Nanoparticles in Cancer in Preclinical
and Clinical Development
•
•
•
•
Carbon nanotubes
Quantum Dots
Nanoparticle-aptamer bioconjugates
Lipid-based nanocarriers
– Liposomes, nanoemulsions, micelles, solid lipid nanoparticles
and nanosuspensions
• Anti-angiogenic molecules in NPs
• Brain-targeted NPs
• Polymeric micelles
• Polymer-drug conjugates and immunoconjugates
• Combination of NPs with other physical and diagnostic methods
– radiotherapy, photodynamic therapy and ultrasound
– nanoshells and paramagnetic NPs
4/8/2013
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Nanoparticle Characterization : NCL/NCI Assay Cascade
http://ncl.cancer.gov/assay_cascade.asp
In Vitro:
Physicochemical:
–Pharmacology
–Size and Shape
–Blood contact
–Composition
properties
–Molecular weight
–Immune cell function
–Surface chemistry
–Cytotoxicity
–Identity
–Mechanistic
–Purity
toxicology
–Stability
–Sterility
–Solubility
In Vivo:
–ADME
–Safety
–Efficacy
Formulation and Process Considerations
• Lessons learned from the marketed nanoparticle drug
products (100 - 1000 nm) that can be applied to the 1-100
nm particles - what is truly new knowledge?
– New processing equipment and characterization methods
– No reference standards and specifications are available
• Need to develop and validate suitable methods and set
meaningful controls and drug product specifications
– establish reference standards for 1-100 nm nanoparticles
• Scale up and manufacturing challenges with acceptable
shelf-life of complex multifunctional nanoparticulate systems
4/8/2013
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Nanoparticle Manufacturing Methods
Homogenization
Top-Down
BULK
Energy
Milling
Cryogenic
Approaches
• Super-Critical Fluid
Technologies
• Spray Freezing into
Liquid
• Ultra-rapid Freezing
4/8/2013
Bottom-Up
NANOPARTICLES
Growth
Precipitation
Emulsion-Diffusion
SOLUTION
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Nanotoxicity : Why the Concern?
• Unusual physicochemical properties attributable to :
– Small size (surface area, size distribution)
• Particle Toxicity Rank (in general): (+) > (-) > (0) (net charge)
– Chemical composition (e.g., purity, crystallinity, electronic
properties)
– Surface structure (e.g., surface reactivity, surface groups,
inorganic or organic coatings)
– Solubility, shape and aggregation
• Opportunities for increased uptake and interaction with
biological tissues relative to bulk materials
• Need to establish nanotoxicity guidelines
• No specific regulations at the present time
– June 9, 2011 FDA Draft Guidance on regulated products involve
Nanotech applications
4/8/2013
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NP Toxicity on Human Lung Cancer A 549 Cells
Choi, S.J. et al; J. Inorg. Biochem. 103: 463-471 (2006)
Layered Metal Hydroxides
(hydrotalcite-type anionic clays)
2 x 104 cells were exposed to NPs (250 and 500 µg/ml) for 72 hrs and then
apoptotic cells were measured by annexin V-FITC (green) binging assay
4/8/2013
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Nanoparticle Pharmacokinetics
• Fate of particles upon administration
– Are all nanoparticles taken up by cells and cross
anatomical barriers ?
– What is their fate after cell uptake?
• Drug absorption, distribution, metabolism and
elimination (ADME):
– How is affected by size, surface composition and
charge of the particles?
• Biopharmaceutical and pharmacokinetic data:
– How to use it to optimize quality and performance of
nanoparticles?
4/8/2013
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Nanomaterial and Nanoparticle ADME Findings
Riviere, J. E. Wiley Wires/Nanomed, Vol 1. 26-34 (2009)
• Most nanomaterials (NMs) accumulate in the liver
– Depending on size and charge, can also accumulate in kidney
and other tissues
• Nanoparticles (NPs) with hydrodynamic radii < 5 - 6 nm
may be eliminated from the kidney
• All classes of NPs have extensive tissue retention
– Carbon based materials and quantum dots - toxicological
implications
• State of NMs once deposited in tissue largely unknown
• Comparisons within and across complex NMs difficult
4/8/2013
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PARENTERAL NANO DRUGS CASE STUDIES
4/8/2013
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Nanoemulsion Stability : TOCOSOL-Paclitaxel
Constantinides, P.P. et. al., Pharm. Res. 17, 175-182, 2000
TOCOSOL
Paclitaxel Loading : 9 mg/ml
Oil
(Vitamin E)
140
Volum e D istribution:
Relative Volume
99% Cumulative Distribution (nm)
Mean Droplet Diameter or
160
120
100
[D ] m ean 4°C
100
80
[D ] m ean 25°C
62 nm
60
40
[D ] 99% 4°C
20
0
10
100
1000
[D ] 99% 25°C
Particle Size (nm )
80
60
Tocophilic
Drug
(Paclitaxel)
Surfactants
(TPGS, P407)
40
0
5
10
15
20
25
30
O
T im e (m o n th s)
O
AcO
R
NH
O
OH
10
6
3'
Paclitaxel potency and levels of degradants were
within specifications throughout the stability study
1'
O
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1
OH
HO
R= C6H5, MW = 853
Solubility < 50 µg/ml
4/8/2013
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O
OAc
O
17
O
Paclitaxel in Blood and Tumor Tissue
P.P. Constantinides, A. Tustian and D.R. Kessler, Adv. Drug Del. Rev. 56 (2004) 1243-1255
Single dose administration of 10 mg/kg to B16 MM-bearing mice
Blood
Tumor
TOCOSOL
Paclitaxel
Taxol®
TOCOSOL
Paclitaxel
Taxol®
8000
AUCTocosol-P = 2.2 AUC
Concentration (ng/g)
Concentration (ng/mL)
10000
8000
6000
4000
Taxol
6000
4000
2000
2000
0
0
0
10
20
30
40
0
Time (hr)
10
20
30
40
Time (hr)
Enhanced TOCOSOL nanoemulsion uptake by tumors due to EPR effect
4/8/2013
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Ibuprofen vs Phospho-Ibuprofen (MDC-917) : Physical
Properties and Cytotoxicity (Nie, T. et al; (2011) British J. Pharmacol.)
MDC : Medicon Pharmaceuticals
PEO-b-PLA diblock copolymer
Soy PC + DSPE-PEG
OH
O
Ibuprofen
209 ± 16 nm
O
O
O
O P
P-I
OCH2CH3
OCH2CH3
-15.5 ± 2.6 mV
Phospho-Ibuprofen
Partition coefficient (log P)
IC50, µM, range
(HT29,HCT116, SW480
colon cancer cell lines)
Cell uptake, nmol/mg
protein
4/8/2013
78 ± 8 nm ; -6.2 ± 0.6 mV
Ibuprofen
Phosphoibuprofen
3.75
5.43
748-1,554
28-104
0.1
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LIPOSOMES
200 nm
MICELLES
50 nm
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In Vitro Cytotoxicity of P-I (MDC-917) Against
Colon Cancer Cell Lines (Nie, T. et al; (2011) British J. Pharmacol.)
A. Empty Nanocarriers
B. Nanocarrier-drug vs Free Drug
P-I
Micellar P-I
Liposomal P-I
200
B
IC50, µM 24- hr
150
100
50
0
SW480
4/8/2013
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HCT116
HT-29
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Antitumor Activity of Liposomal P-I (MDC-917) in
Human Colon Cancer (SW 840)-Bearing Mice
(Nie, T. et al; (2011) British J. Pharmacol.)
0.8
Dose : 100 mg/kg/day i.p.
Control
#
Tumor Volume, mm3
P-I
#
*
(Mean ± SEM, n=20)
#
#
Tumor weight, g
600
Liposomal P-I
400
200
0.6
0.4
#
#
0.2
0
0
5
10
15
20
25
Treatment, days
0.0
Vehicle control
4/8/2013
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P-I
Liposomal P-I
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ORAL NANO DRUGS CASE STUDIES
4/8/2013
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Tamoxifen Citrate
VAN Life Sciences Pvt Ltd, www.van.in
Log PO/W = 3.7, pH 7.0
BCS IV Molecule
• Oral anti-estrogen for breast cancer treatment
• Available as a Tablet and Oral Solution in a daily dose 10-20 mg;
chronic therapy (3-5 yrs)
• Hepatotoxicity is a major toxicity with TMX-Citrate therapy
• Poor oral bioavailability (20-30%); large inter-subject variability
• Intestinal P-gp substrate; First-pass metabolism (CYP34A)
• Use of CYP34A inhibitors improves bioavailability
• Use of lipid-based systems (SNEDDS, SLN/NLC) to
improve the oral BA of TMX
4/8/2013
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Pharmacokinetics of Tamoxifen in Fasted Rats
VAV Life Sciences, Pvt, Ltd, www.vav.in
Formulation
Cmax (ng/ml)
Tmax (h)
AUC0-∞(ng/ml-h)
t1/2 (h)
TMX-SNEDDS
680.12±55.54
2.0
9873.031
6.58
TMX citrate
solution
Commercial
formulation
275.54±25.34
2.0
2628.71
4.77
TMX base
75.33±12.34
4.0
1100.31
11.59
1 4-fold bioavailability enhancement compared to TMX-citrate and 9-fold
enhancement compared to TMX free base (TMX Dose : 10 mg/kg)
Formulation is physically and chemically stable at room temperature for
at least 6 months; Formulation is stable in simulated GI fluids for 8 hr.
4/8/2013
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Antitumor Efficacy in DMBA-Induced Breast-Tumor Bearing Rats
% Tumor Volume
VAV Life Sciences, Pvt, Ltd, www. vav.in
3 mg/kg every 3 days for 30 days
700
Control
600
Tamoxifen citrate
a***
Tamoxifen SMEDDS
SEDDS
500
400
**p< 0.01; *** p< 0.001
300
Mean ± SD, n=5
b***
a**
200
100
0
0
5
10
15
20
25
30
35
Days
4/8/2013
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Effect of Oral Tamoxifen on Liver Toxicity Markers
VAV Life Sciences, Pvt, Ltd, www.vav.in
(A)
80
a*
70
60
AST (IU/L)
(B)
b*
50
40
30
20
10
Control
a***
70
b***
60
ALT (IU/L)
80
50
40
30
20
10
0
Tmx-Citrate
0
Control
Tmx Citrate
Tmx SEDDS
Control
Tmx Citrate
Tmx SEDDS
hepato-toxicity markers (mean ± SD, n=6) of rats
treated with 3 mg/kg every 3 days for 30 days
Lipid Peroxidation (MDA)
nm/mg of protein
70
(C)
60
a***
50
Parenchymal
degeneration,
lymphocyte
infiltration,
and cell
apoptosis
b***
40
Tmx-SNEDDS
30
20
10
0
Control
Tmx Citrate
Tmx SEDDS
AST : Aspartate Transaminase
ALT : Alanine Transaminase
4/8/2013
Liver histopathology after 30-day
treatment
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Peroral Absorption of Leuprolide in Dogs in EC HGCs
Constantinides P.P. et al. 2002 AAPS Meeting and Exposition, Toronto, Canada
Formulation
n
Dose
mg/k
g
LPM EC (2X)
5
2.09
31277.2 ± 9413
199.2 ± 78.5
11.6 ± 3.7
LPM EC (4X)
6
1.04
16158.7 ± 3093
117.9 ± 20.3
11.4 ± 2.2
LPM EC (2X)
6
0.35
8563 ± 1535
81.5 ± 16.8
16.6 ± 3.4
LPM EC (2X)
3
0.35
6208 ± 2311
68.3 ± 24.4
13.8 ± 5.1
LPM EC (4X)
3
0.35
7488.3 ± 2609
39.2 ± 15.0
16.6 ± 5.8
AUC
(min*ng/ml)
Cmax
(ng/ml)
Bioavailability
(mean  SEM)
Leuprolide Plasma Concentration
(ng/ml, mean ± SEM)
400
EC : Enteric Coated; X is the number of layers of coating
applied: HGC : Hard Gelatin Capsule (LiCaps®)
Oral (i.d.) BA of Leurpolide from solution = 2.2 ± 0.2 (n=4)
350
300
250
200
Reverse Micelle
150
2.09
1.04
0.35
0.35
0.35
100
50
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
EC
EC
EC
EC
EC
(2X)
(4X)
(2X)
(2X)
(4X)
Solubility in
0.1 M acetate
buffer pH 5.1
> 30 mg/ml
Intestinal
permeability:
very low
Leuprolide (LHRH
analogue, MW = 1209)
p-Glu-His-Trp-SerTyr-D-Leu-Leu-ArgPro-NHC2H5
•
•
Prostate Cancer
•
Precocious Puberty
Endometriosis
0
0
100
200
300
400
500
600
Time (min)
4/8/2013
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Conclusions and Future Perspectives
• There have been significant and promising advances in drug
delivery NPs in cancer drug delivery and targeting.
• The most promising systems/approaches are those that
combine disease diagnosis with therapy (Nanotheranostics).
– Formulation development and manufacturing challenges
• Better understanding of the ADMET and PK of NPs is critical to
their progress from bench to clinic and commercialization
• Toxicity is dependent on the nature and composition of NPs
– In general, inorganic NPs are more toxic than organic ones
– Need to establish nanotoxicity guidelines
• Expanded use of parenteral and oral lipidic and/or polymeric
nanoparticles in cancer drug delivery for both poorly soluble and
water-soluble molecules/macromolecules.
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Acknowledgements
• Nanoemulsions : TOCOSOL-Paclitaxel
– Karl Lambert, Alex Tustian, Dean Kessler, former R&D
associates at SONUS Pharmaceuticals
• Liposomes/Polymeric Micelles : Phospho-Ibuprofen
– Collaboration with Dr. Basil Rigas and laboratory staff,
Stony Brook University, Medical School, Division of
Cancer Prevention and Medicon Pharmaceuticals
• LPM™ (Reverse Micelles) : Leuprolide (DOR Biopharma)
– Andy Jang, Likan Liang, Dave Fast, Liangxiu He, former
R&D associates at DOR Biopharma
• Tamoxifen-SNEDDS
– Arun Kedia, General Manager of VAV Life Sciences Pvt,
Ltd, Mumbai, INDIA, a partnering company
4/8/2013
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ENABLED ANTICANCER DRUG PRODUCTS
THANK YOU !
LIPIDS/POLYMERS
ORGANIC/INORGANIC MATERIALS
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FUNCTIONAL
NANOPARTICLES
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