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By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen

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

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By Michael A. Sandoval Dr. Zhengrong Cui Dr. J. Mark Christensen

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  1. 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

  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 Gemcitabine Stearic Acid GemC18

  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 GemC18

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

  19. Nanoparticle Formulation Heat Add H2O Add Cool to Room T. Surfactant Lecithin and other lipids Slurry Warm emulsion Solid lipid NPs in suspension Potential Delivery NP Slurry Warm emulsion Solid lipid NPs in suspension

  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 NP Nanoparticles Prodrug and NP conjugation “GemC18”

  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 G G G G G G G G NP G G G G G G G G GemC18 in NPs G Gemcitabine G G G G G G M G G GemC18 in Micelles

  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 MTT Formazan

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

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

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

  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

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