Superior Anti-tumor Activity From A
Gemcitabine Prodrug Incorporated Into
Lecithin-Based Nanoparticles
By
Michael A. Sandoval
Dr. Zhengrong Cui
Dr. J. Mark Christensen
Department of Pharmaceutical Sciences
Why Research?


Leading causes of death in U.S
Undesirable clinical side effects of therapeutic
drugs

Efforts to develop superior delivery methods

Improve drug circulation
http://www.brighamandwomens.org/publicaffairs/Images/Pill_bottle_and_pills.jpg
Cancer Perspective

Leading cause of death in U.S

1.4 million new cases in 2007; 2009?

$2.3 billion dollars in 2005;

~1,500 daily mortality
2009?
Cancer Overview

Not a novel disease (1500 B.C)

Disease of uncontrollable cell division

An array of unknown causes

All age groups susceptible

85% cancers relate to solid tumors
Cancer Treatment (Tx)

Chemotherapy (1940) and radiotherapy (N.C.T)

Chemotherapy drugs fall into 2 categories (cell cycle)

Tx efficacy is dependent on time

No single “cure for cancer”

Undesirable side effects (alopacia, nausea, susceptibility)
Gemcitabine Hydrochloride

Eli Lilly & Company

Most important drug since Ara C (1969)

Approved by F.D.A in 2004

Given through infusion (i.v.)
Gemcitabine Pharmacology

Difluorodeoxycytidine (dFdCyd)

Belongs to group of antimetabolites (specific)

Undergoes intracellular metabolism
 Blood,

liver, and kidneys
Half-life of 8-17 min
Gem. Pharmacology Continued

Analogue of deoxycytidine nuceloside

Cell cycle specific
 G0,
G1, S, G2, and M Phase
Nucleoside Transporters
Gemcitabine Mechanism
Gemcitabine Application

Chemotherapeutic Agent

Treat various types of cancer
 Non
Small Cell Lung Cancer*
 Pancreatic Cancer
 Metastatic Breast Cancer*
 Ovarian cancer*
*Combination Therapy
Non Small Cell Lung
Cancer
Pancreatic Cancer
Metastic Breast Cancer
Ovarian Cancer
Gemcitabine Inadequacy

Short half-life

Rapid metabolism

Toxicity
 Clinical
Table 1: Gemcitabine Half-Life For “Typical” Patient
side effects
 High doses to achieve therapeutic benefit
Why Inadequate?
Cancer Incidence Rates
Overcoming Gemcitabine’s Limitations

Goal: To improve in vivo anti-tumor activity of
gemcitabine
Our Strategy
 Prodrug synthesis
 Clearance

time
Nanoparticle incorporation
 Delivery
 Specificity
Synthesis of Prodrug

Reaction synthesis of “GemC18”

Stearic acid (F.A) addition
Gemcitabine
Stearic Acid
GemC18
Why Use A Prodrug?
Administered in an inactive form
 A.D.M.E optimization
 Bioavailability & Selectivity

https://www.dnadirect.com/img/content_images/resources/genes_and_drugs/proVsActiveDrug.gif
GemC18 Characterization

Thin layer chromatography (TLC)
GemC18

Nuclear magnetic resonance (NMR)
GemC18
GemC18 Purification
Nitrogen+Solvent


‘Flash’ silica gel column
Separate non-conjugated S.A
Sample
Sand
Silica gel
x24 Culture Tube
Nanoparticle Formulation
Heat
Add H2O
Lecithin and other
lipids
Add
Cool to
Surfactant
Room T.
Slurry
Warm emulsion
Solid lipid NPs
in suspension
Potential Delivery
NP
Slurry
Warm emulsion
Solid lipid NPs
in suspension
NP Formulation Cont.
TEM=Transmission Electron
Microscope
~180 nm diameter
Surfactant Concentration
Why Use Nanoparticles?

Delivery system for small molecules/macro

Enhance solubility of poorly water soluble drugs


Can be engineered to prevent RE system uptake
and improve targeting
Improve drug stability
Incorporation of GemC18 Into NPs


GemC18 is now lipophilic
Gem. on surface of NP
NP
“GemC18”
Nanoparticles
Prodrug and NP conjugation
Change in NP Size
200
Particle Size (nm)
190
180
170
160
150
Blank NP
GemC18 NP
GemC18 Incorporated Into NPs
100 µg GemC18-NPs
NPs alone
GemC18 micelles
0.12
0.11
0.6
GemC18 (OD248)
0.10
0.09
0.4
0.08
0.07
0.2
0.06
0.0
0.05
0
5
10
15
Fraction (0.25 ml)
20
GemC18 micelles (OD248)
0.8
Gel Permeation Chromatography

Separation based on molecular size

Confirmation of GemC18-NP

Sepharose 4b (resin)

No micelle peaks
Desired Sample
5mg/ml Of GemC18 Into NPs
GemC18 (OD248)
4
5 mg GemC18-NPs
1 mg GemC18-NPs
500 µg GemC18-NPs
GemC18 micelles
3
2
1
0
0
5
10
15
Fraction (0.25 ml)
20
Release Of GemC18 From NPs
100
% GemC18 released
80
60
40
GemC18 in micelles
20
GemC18 NPs
0
0
0.5% SDS in PBS
release medium
50
100
150
Time (min)
200
250
Release Study Expansion
G
G
G
G G G
G
NP
G
G
G
G
G G
M
G
G
G
G
G
G
G
GemC18 in
NPs
G Gemcitabine
G
G
G G
GemC18 in
Micelles
GemC18-NP In Culture
60
TC-1 LC50 (pM)
50
24 hours
48 hours
40
30
20
10
0
Gemcitabine
GemC18-NP
GemC18-NP PEG
PEG = Poly Ethylene Glycol
TC1= Mouse Lung Cancer Cells
Cell Viability Assay
Measures activity of mitochondrial enzymes
 MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide
 Measures cell viability
 Quantification by measuring wavelength @
590 nm

MTT
Formazan
Why Use Polyethylene Glycol?


Polymer, low toxicity, abundant
PEG improves drug circulation (reticuloendothelial
system)
NP
NP
Prodrug and NP conjugation
PEG
Prodrug Incorporated into
NP, plus PEG
PanC02 Cytotoxicity Assay
100
PanCO2 LC50 (pM)
80
24 hours
48 hours
60
40
20
0
Gemcitabine
GemC18-NP
GemC18-NP PEG
PanC02 = Mouse Pancreatic Cancer Cell Line
GemC18-NP Were Toxic To BxPC3
48 hours
Log(Fa/Fu)
0.5
-3
-2
-1
0
-0.5
-1.5
Gemcitabine
GemC18-NP
GemC18-NP PEG
Log[Dose]
The BxPC3 is a human pancreatic cancer cell line
In vitro Data Summary
In mouse cancer lines:

 GemC18-NP
less toxic than Gem after 24 hours
 After 48 hrs, GemC18-NP much more toxic
GemC18-NP toxicity takes longer to take place

60
PanCO2 LC50 (pM)
TC-1 LC50 (pM)
80
40
30
20
24 hours
48 hours
0.5
Log(Fa/Fu)
50
100
24 hours
48 hours
60
40
-3
0
0
Gemcitabine
GemC18-NP
GemC18-NP PEG
-1.5
Gemcitabine
GemC18-NP
GemC18-NP PEG
-1
-0.5
20
10
-2
Gemcitabine
GemC18-NP
GemC18-NP PEG
Log[Dose]
0
Mice Tumor Implantation



C57BL/6 mice (n = 6-7)
 TC-1 Cells (mouse lung cancer)
Subcutaneous (s.c) administration of tumor
 Mouse lung cancer
 Day 0
 Day 4
I.v injection of drug
Antitumor Mouse Efficacy Study
Tumor diameter (mm)
9
UN
Gem i.v.
Gem i.p.
GemNP i.v.
6
3
0
5
10
15
Time (d.p.i.)
20
TC-1 model lung cancer in C57BL/6 mice (n = 6-7)
Gem: 94 mMoles/kg for the i.v. route
380 mMoles/kg for the i.p. route (= 100 mg/kg)
Percent Tumor-bearing Mice
100
% Tumor free mice
80
60
40
UN
Gem i.v.
Gem i.p.
GemNP i.v.
20
0
5
10
15
Time (d.p.i.)
20
25
Advanced Tumor Study
Tumor diameter (mm)
14
GemC18-NP
Un
12
10
8
6
0
2
4
6
Time (days post treatment)
8
Conclusions




Average nanoparticles size was 180 nm
GemC18 prodrug was incorporated into NPs at a
maximum concentration of 5mg/ml
GemC18 in the NPs was toxic to tumor cells
GemC18 NPs are far more superior than native
gemcitabine in mouse efficacy study
Acknowledgements
‣ Dr. Zhengrong Cui
‣ Nija Yan
‣ Letty Rodriguez
‣ Yu Zhen
‣ Xiran Li
‣ Woongye Chung
‣ Dr. J. Mark Christensen
‣ Dr. Phil Proteau
‣ Dong Li
‣ Dr. Alex Chang