1. By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen Department of Pharmaceutical Sciences Superior Anti-tumor Activity From A Gemcitabine Prodrug Incorporated Into Lecithin-Based Nanoparticles

2. 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

3. 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

4. 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

5. 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)

6. Gemcitabine Hydrochloride  Eli Lilly & Company  Most important drug since Ara C (1969)  Approved by F.D.A in 2004  Given through infusion (i.v.)

7. Gemcitabine Pharmacology  Difluorodeoxycytidine (dFdCyd)  Belongs to group of antimetabolites (specific)  Undergoes intracellular metabolism  Blood, liver, and kidneys  Half-life of 8-17 min

8. Gem. Pharmacology Continued  Analogue of deoxycytidine nuceloside  Cell cycle specific  G0, G1, S, G2, and M Phase Nucleoside Transporters

9. Gemcitabine Mechanism

10. Gemcitabine Application  Chemotherapeutic Agent  Treat various types of cancer  Non Small Cell Lung Cancer*  Pancreatic Cancer  Metastatic Breast Cancer*  Ovarian cancer* * Combination Therapy

11. 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

12. Why Inadequate?

13. Cancer Incidence Rates

14. Overcoming Gemcitabine’s Limitations  Goal: To improve in vivo anti-tumor activity of gemcitabine Our Strategy  Prodrug synthesis  Clearance time  Nanoparticle incorporation  Delivery  Specificity

15. Synthesis of Prodrug  Reaction synthesis of “GemC18”  Stearic acid (F.A) addition GemcitabineStearic AcidGemC18

16. 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

17. GemC18 Characterization  Thin layer chromatography (TLC)  Nuclear magnetic resonance (NMR) GemC18

18. GemC18 Purification  ‘Flash’ silica gel column  Separate non-conjugated S.A Sand Silica gel x24 Culture Tubes Sample Nitrogen+Solvent

19. Nanoparticle Formulation Heat Add H 2 O 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

20. NP Formulation Cont. TEM=Transmission Electron Microscope ~180 nm diameter Surfactant Concentration

21. 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

22. Incorporation of GemC18 Into NPs  GemC18 is now lipophilic  Gem. on surface of NP “GemC18” NanoparticlesProdrug and NP conjugation NP

23. Change in NP Size

24. GemC18 Incorporated Into NPs

25. Gel Permeation Chromatography  Separation based on molecular size  Confirmation of GemC18-NP  Sepharose 4b (resin)  No micelle peaks Desired Sample

26. 5mg/ml Of GemC18 Into NPs

27. Release Of GemC18 From NPs 0.5% SDS in PBS release medium

28. Release Study Expansion GemC18 in NPs G G G G G G G G G G G G G G G G NP G G G G G G G G G M GemC18 in Micelles Gemcitabine

29. GemC18-NP In Culture PEG = Poly Ethylene Glycol TC1= Mouse Lung Cancer Cells

30. 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 MTTFormazan

31. Why Use Polyethylene Glycol?  Polymer, low toxicity, abundant  PEG improves drug circulation (reticuloendothelial system) NP Prodrug and NP conjugationPEG NP Prodrug Incorporated into NP, plus PEG

32. PanC02 Cytotoxicity Assay PanC02 = Mouse Pancreatic Cancer Cell Line

33. GemC18-NP Were Toxic To BxPC3 The BxPC3 is a human pancreatic cancer cell line 48 hours

34. 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

35. 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

36. Antitumor Mouse Efficacy Study 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)

37. Percent Tumor-bearing Mice

38. Advanced Tumor Study

39. 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

40. 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