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![Page 1: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/1.jpg)
By
Michael A. SandovalDr. Zhengrong Cui
Dr. J. Mark Christensen
Department of Pharmaceutical Sciences
Superior Anti-tumor Activity From A Gemcitabine Prodrug
Incorporated Into Lecithin-Based Nanoparticles
![Page 2: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/2.jpg)
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
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Cancer Perspective
Leading cause of death in U.S
1.4 million new cases in 2007; 2009?
$2.3 billion dollars in 2005; 2009?
~1,500 daily mortality
![Page 4: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/4.jpg)
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
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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)
![Page 6: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/6.jpg)
Gemcitabine Hydrochloride
Eli Lilly & Company
Most important drug since Ara C (1969)
Approved by F.D.A in 2004
Given through infusion (i.v.)
![Page 7: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/7.jpg)
Gemcitabine Pharmacology
Difluorodeoxycytidine (dFdCyd)
Belongs to group of antimetabolites (specific)
Undergoes intracellular metabolism Blood, liver, and kidneys
Half-life of 8-17 min
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Gem. Pharmacology Continued Analogue of deoxycytidine nuceloside
Cell cycle specific G0, G1, S, G2, and M Phase
Nucleoside Transporters
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Gemcitabine Mechanism
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Gemcitabine Application
Chemotherapeutic Agent
Treat various types of cancer Non Small Cell Lung Cancer* Pancreatic Cancer Metastatic Breast Cancer* Ovarian cancer*
*Combination Therapy
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Gemcitabine Inadequacy
Short half-life
Rapid metabolism
Toxicity Clinical side effects High doses to achieve therapeutic benefit
Table 1: Gemcitabine Half-Life For “Typical” Patient
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Why Inadequate?
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Cancer Incidence Rates
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Overcoming Gemcitabine’s Limitations Goal: To improve in vivo anti-tumor
activity of gemcitabine
Our Strategy Prodrug synthesis
Clearance time Nanoparticle incorporation
Delivery Specificity
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Synthesis of Prodrug
Reaction synthesis of “GemC18”
Stearic acid (F.A) addition
Gemcitabine Stearic Acid GemC18
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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
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GemC18 Characterization
Thin layer chromatography (TLC)
Nuclear magnetic resonance (NMR)
GemC18
GemC18
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GemC18 Purification
‘Flash’ silica gel column Separate non-conjugated S.A
Sand
Silica gel
x24 Culture Tubes
Sample
Nitrogen+Solvent
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Nanoparticle Formulation
Heat Add H2O
Slurry
Surfactant
Add
Warm emulsion
Cool to
Room T.
Solid lipid NPs in suspension
Lecithin and other lipids
Slurry Warm emulsion Solid lipid NPs in suspension
NP
Potential Delivery
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NP Formulation Cont.
TEM=Transmission Electron Microscope
~180 nm diameter
Surfactant Concentration
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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
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Incorporation of GemC18 Into NPs GemC18 is now lipophilic Gem. on surface of NP
“GemC18” Nanoparticles Prodrug and NP conjugation
NP
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Change in NP Size
150
160
170
180
190
200
Blank NP GemC18 NP
Par
ticl
e S
ize
(nm
)
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GemC18 Incorporated Into NPs
0.0
0.2
0.4
0.6
0.8
0 5 10 15 20Fraction (0.25 ml)
Gem
C18
(O
D24
8)
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
Gem
C18
mic
elle
s (O
D24
8)
100 µg GemC18-NPsNPs aloneGemC18 micelles
![Page 25: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/25.jpg)
Gel Permeation Chromatography Separation based on molecular size Confirmation of GemC18-NP
Sepharose 4b (resin)
No micelle peaksDesired Sample
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5mg/ml Of GemC18 Into NPs
0
1
2
3
4
0 5 10 15 20Fraction (0.25 ml)
Gem
C18
(O
D24
8)5 mg GemC18-NPs1 mg GemC18-NPs500 µg GemC18-NPsGemC18 micelles
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Release Of GemC18 From NPs
0
20
40
60
80
100
0 50 100 150 200 250Time (min)
% G
em
C1
8 r
ele
as
ed
GemC18 in micelles
GemC18 NPs
0.5% SDS in PBS release medium
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Release Study Expansion
GemC18 in NPs
G
GG
G
G
G
G
G
G
G
G
G
GG
GG
NP
G G
G
G G
G G
G
G
M
GemC18 in Micelles
Gemcitabine
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GemC18-NP In Culture
0
10
20
30
40
50
60
Gemcitabine GemC18-NP GemC18-NP PEG
TC
-1 L
C50
(p
M)
24 hours
48 hours
PEG = Poly Ethylene GlycolTC1= Mouse Lung Cancer Cells
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Cell Viability Assay
Measures activity of mitochondrial enzymes
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Measures cell viability Quantification by measuring
wavelength @ 590 nm
MTT Formazan
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Why Use Polyethylene Glycol?
Polymer, low toxicity, abundant PEG improves drug circulation
(reticuloendothelial system)
NP
Prodrug and NP conjugation
PEG
NP
Prodrug Incorporated into NP, plus PEG
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PanC02 Cytotoxicity Assay
0
20
40
60
80
100
Gemcitabine GemC18-NP GemC18-NP PEG
Pan
CO
2 LC
50 (
pM)
24 hours
48 hours
PanC02 = Mouse Pancreatic Cancer Cell Line
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GemC18-NP Were Toxic To BxPC3
-1.5
-0.5
0.5
-3 -2 -1 0
Log[Dose]
Log(
Fa/F
u)
Gemcitabine
GemC18-NP
GemC18-NP PEG
The BxPC3 is a human pancreatic cancer cell line
48 hours
![Page 34: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/34.jpg)
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
0
10
20
30
40
50
60
Gemcitabine GemC18-NP GemC18-NP PEG
TC
-1 L
C50
(p
M)
24 hours
48 hours
0
20
40
60
80
100
Gemcitabine GemC18-NP GemC18-NP PEG
Pan
CO
2 LC
50 (
pM)
24 hours
48 hours
-1.5
-0.5
0.5
-3 -2 -1 0
Log[Dose]
Log(
Fa/F
u)
Gemcitabine
GemC18-NP
GemC18-NP PEG
![Page 35: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/35.jpg)
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
![Page 36: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/36.jpg)
Antitumor Mouse Efficacy Study
0
3
6
9
5 10 15 20Time (d.p.i.)
Tu
mo
r d
iam
ete
r (m
m)
UNGem i.v.Gem i.p.GemNP i.v.
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)
![Page 37: By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine.](https://reader035.fdocuments.us/reader035/viewer/2022062717/56649e2b5503460f94b19a37/html5/thumbnails/37.jpg)
Percent Tumor-bearing Mice
0
20
40
60
80
100
5 10 15 20 25Time (d.p.i.)
% T
um
or
free m
ice
UNGem i.v.Gem i.p.GemNP i.v.
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Advanced Tumor Study
6
8
10
12
14
0 2 4 6 8
Time (days post treatment)
Tu
mo
r d
iam
ete
r (m
m)
GemC18-NP
Un
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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
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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