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

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; 2009?

~1,500 daily mortality

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

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

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)

Nuclear magnetic resonance (NMR)

GemC18

GemC18

GemC18 Purification

‘Flash’ silica gel column Separate non-conjugated S.A

Sand

Silica gel

x24 Culture Tubes

Sample

Nitrogen+Solvent

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

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

“GemC18” Nanoparticles Prodrug and NP conjugation

NP

Change in NP Size

150

160

170

180

190

200

Blank NP GemC18 NP

Par

ticl

e S

ize

(nm

)

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

Gel Permeation Chromatography Separation based on molecular size Confirmation of GemC18-NP

Sepharose 4b (resin)

No micelle peaksDesired Sample

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

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

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

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

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

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

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

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

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

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

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)

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.

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

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