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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