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Ain shams Universirty Faculty Of Science Evaluation of anticancer effects of a novel proteasome inhibitor (Velcade), interferon (alpha-interferon) and antimyeloma antibodies on the growth of myeloma cells. Thesis Submitted for the Requirement of the Ph.D degree in Biochemistry By Hanan Mohammed Mohammed El shershaby. (M.Sc in Biochemistry) Under Supervision of Prof.Dr / Ibrahim Hassan Borai Prof.Dr/ Mohamed Taha Hussein El- Kolaly Prof. of Biochemistry Professor of Radiopharmaceutics Faculty Of Science Atomic Energy Authority Ain shams Universirty Prof.Dr/Naser Mohammed El-lahloby Prof. Dr/ Kamel Abd El.hameed Moustafa Prof. of Medical Oncology Professor of Biochemistry Cancer Institute Center Atomic Energy Authority Cairo University Dr / Ahmed Abd El.aziz sayed Assistant professor of Biochemistry Faculty Of Science Ain shams Universirty (2013) CONTENTS Introduction and Aim of the work 1 Review Of Literature 4 ◙ Multiple Myeloma 4 ◙ Cell cycle 16 ◙ Apoptosis 19 ◙ Caspases 23 ◙ Chemotherapy 25 ◙ Proteasomes 36 Bortezomib (Velcade) 38 ◙ Interferons 43 ◙ Immunity and Immune system 46 Antibodies 49 Production of Antibodies 51 ◙ Immunotherapy 57 ◙ Radioimmunotherapy ( RIT ) 61 ◙ Gene therapy 62 Materials and Methods 64 Results 92 Discussion 133 Summary 144 References 148 Arabic Summary LIST OF FIGURES Figure (1): Overview of the cell cycle. 16 Figure (2): Apoptosis – the programmed death of a cell. 20 Figure (3): The intrinsic and extrinsic pathways leading to apoptosis. 22 Figure (4): Structure of the 26S proteasome. 37 Figure (5): Chemical Structure of Bortezomib 38 Figure (6): Mechanism of action of Bortezomib 40 Figure (7): The different ways that Bortezomib can affect multiple myeloma 41 Figure (8): Antitumor actions of interferons. 45 Figure (9): The principal mechanism of innate and adaptive immunity. 48 Figure (10): General structure of Antibodies. 50 Figure (11): Antibody therapeutics. Mechanisms through which antitumor antibody can mediate tumor cell killing directly (ADCC, Opsonization, complement activation) or indirectly through delivery of acytotoxic radionuclide, drug, or other toxin to tumor) 60 Figure (12): Radioimmunotherapy (RIT) of Cancer 62 Figure (13): Effect of variable doses of Bortezomib on the viability % of myeloma cells 93 Figure (14): Effect of variable doses of IFN-α on the viability % of myeloma cells. 94 Figure (15): Effect of variable doses of combined treatment with Bortezomib and IFN-α on the viability % of myeloma cells. 95 Figure (16): Effect of variable doses of Myeloma Antibodies on the viability % of myeloma cells. 96 Figure (17): Effect of variable doses of Labeled Myeloma- Antibodies on the viability % of myeloma cells. 97 Figure (18): Effect of variable doses of combined treatment with bortezomib + Alpha-Interferon+ Myeloma-Antibodies on the viability % of myeloma cells. 98 Figure (19): Effect of variable doses of Bortezomib on viability% of myeloma cells of ascites bearing mice (mean ± SD). 101 Figure (20): Effect of variable doses of IFN-α on viability % of myeloma cells of ascites bearing mice (mean ± SD). 103 Figure (21): Effect of variable doses of combined treatment with Bortezomib + IFN-α on the viability % of myeloma cells of ascites bearing mice (mean ± SD). 105 Figure (22): Effect of variable doses of Myeloma-Antibodies on viability % of myeloma cells of ascites bearing mice (mean ± SD). 107 Figure (23): Effect of variable doses of Labeled myeloma- Antibodies on viability % of myeloma cells of ascites bearing mice (mean ± SD). 109 Figure (24): Effect of variable doses of combined treatment with Bortezomib + IFN-α +Myeloma-Antibodies on the viability % of myeloma cells of ascites bearing mice (mean ± SD). 111 Figure (25): ELISA results of binding of antimyeloma antibodies to myeloma cells. 112 Figure (26): Elution pattern of radioiodinated mixture of myeloma antibodies (Rabbit (1)) and purification of 125 I- antibodies tracer using PD-10 column (Ch-T method). 113 Figure (27): Elution pattern of radioiodinated mixture of myeloma antibodies (Rabbit (2)) and purification of 125 I- antibodies tracer using PD-10 column (Ch-T method). 114 Figure (28): Elution pattern of radioiodinated mixture of myeloma antibodies (Rabbit (3)) and purification of 125 I- antibodies tracer using PD-10 column (Ch-T method). 115 Figure (29): Elution pattern of radioiodinated mixture of myeloma antibodies (Rabbit (4)) and purification of 125 I- antibodies tracer using PD-10 column (Ch-T method). 116 Figure (30): Elution pattern of radioiodinated mixture of polying myeloma antibodies and purification of 125 I-antibodies tracer using PD-10 column (Ch-T method). 117 Figure (31): Electrophoresis analysis of 125I-antibodies. 118 Figure (32): Flow cytometric analysis of cell cycle profile of myeloma cell line (SP2OR) without treatment [control]. 119 Figure (33):Flow cytometric analysis for the effect of Bortezomib on cell cycle profile of myeloma cell line (SP2OR). 120 Figure (34): Flow cytometric analysis for the effect of IFN-α on cell cycle profile of myeloma cell line (SP2OR). 121 Figure (35): Flow cytometric analysis for the effect of Bortezomib+ IFN-α on cell cycle profile of myeloma cell line (SP2OR). 122 Figure (36): Flow cytometric analysis for the effect of Myeloma- antibodies on cell cycle profile of myeloma cell line (SP2OR). 123 Figure (37): Flow cytometric analysis for the effect of Labeled myeloma antibodies on cell cycle profile of myeloma cell line (SP2OR). 124 Figure (38): Flow cytometric analysis for the effect of Bortezomib+ IFN-α + Myeloma-antibodies on cell cycle profile of myeloma cell line (SP2OR). 125 LIST OF TABLES Table (1): Schema of Pathophysiology. 8 Table (2): Durie and Salmon Staging System. 9 Table (3): International Staging System (ISS). 10 Table (4): Diagnosis in Multiple Myeloma. 11 Table (5): Myeloma Treatment Options. 14 Table (6): Some drugs used in treatment of myeloma. 15 Table (7): Cell cycle phases. 17 Table (8): Subfamily members of caspase family. 24 Table (9): Possible Side Effects of Bortezomib (Velcade). 42 Table (10): Possible mechanisms for IFNs antitumour action in myeloma. 44 Table ( 11 ): Classification of Cancer immunotherapy. 59 Table (12): Total count and viability % of myeloma cells of untreated and treated ascites bearing mice with Bortezomib. 100 Table (13): Total count and viability % of myeloma cells of untreated and treated ascites bearing mice with Alpha- interferon. 102 Table (14): Total count and viability % of myeloma cells of untreated and treated ascites bearing mice with Bortezomib plus α-interferon. 104 Table (15): Total count and viability % of myeloma cells of untreated and treated ascites bearing mice with Myeloma- antibodies. 106 Table (16): Total count and viability % of myeloma cells of untreated and treated ascites bearing mice with Labeled myeloma antibodies. 108 Table (17): Total count and viability % of myeloma cells of untreated and treated ascites bearing mice with Bortezomib + α- interferon+ Myeloma-Antibodies. 110 Table (18) Effects of Bortezomib, α-interferon, (Bortezomib + α- interferon), Myeloma-Antibodies, Labeled myeloma antibodies. and (Bortezomib + α-interferon+ Myeloma-Antibodies) on Cell Cycle Phase Distribution. 126 Table (19): The weight of ascites bearing mice before and after treatment (mean ± SD). 127 Table (20): The level of β2-microglobulin in Myeloma cells treated with Bortezomib, IFN-α , Bortezomib+ IFN-α , Myeloma-antibodies, Labeled myeloma antibodies and Bortezomib+ IFN-α +Myeloma-antibodies compared that of control (Mean±SD). 128 Table (21): The Activities of caspase-8 and caspase- 9 in Myeloma cells treated Bortezomib, IFN-α , Bortezomib+ IFN-α , Myeloma-antibodies, Labeled myeloma antibodies and Bortezomib+ IFN-α +Myeloma-antibodies compared that of control (Mean±SD). 129 Table (22): Change in liver marker enzymes ALT, AST and ALP in Ascites bearing group and treated groups compared to normal control (mean± SD). 130 Table (23) Change in Hemoglobin (Hb) and Kidney functions in Ascites bearing group and treated groups compared to normal control (mean± SD). 132 LIST OF ABBREVIATIONS Ab : Antibody ADCC : Antibody-dependent cellular cytotoxicity AIF : Apoptosis inducing factor Apaf-1 : Apoptotic protease activating factor 1 ATP : Adenosine triphosphate BAX : BCL2-associated × protein BID : BH3-interacting domain death agonist BRM : Biological response modifier Bz : Bortezomib Ch-T : Chloramine –T Ci : Curie CR : A complete response CRP : C-reactive protein CTLs : Cytotoxic T lymphocytes D : Dose DC : Dendritic cells DISC : Death-inducing signal complex DMSO : Dimethyle sulfoxide DTT : Dithiothreitol FADD : Fas-associated death domain protein FasL : Fas ligand FBS : Foetal bovine serum FCA : Freund’s complete adjuvant FIA : Freund’s incomplete adjuvant 5-FU : 5-fluorouracil G0 : Gap 0 G1 : Gap 1 G2 : Gap 2 HDC : High-dose chemotherapy HSCT : Hematopoietic stem cell transplant IAP : Inhibitor of apoptosis IFNs : Interferons IFN-α : Alpha-Interferon ISS : The International Staging System KI : Potassium iodide Mo : Monoclonal protein M : Mitosis MAbs : Monoclonal antibodies MGUS : Monoclonal gammopathy of undetermined significance MHC : Major histocompatibility complex MIP-1α : Macrophage inflammatory protein-1α MM : Multiple myeloma 6-MP : 6-mercaptopurine Na 125 I : Sodium Iodide-125 Na OH : Sodium Hydroxide Na 2HPO 4.2H2O: Di-Sodium hydrogen orthophosphate Na 2S2O5 : Sodium metabisulphite NaCl : Sodium chloride NaH 2PO 4.2H2O: Sodium dihydrogen orthophosphate: NaHCo 3 : Sodium bicarbonate NK : Natural killer
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  • Velcade, INN-Bortezomib

    Velcade, INN-Bortezomib

    SCIENTIFIC DISCUSSION This module reflects the initial scientific discussion for the approval of Velcade. This scientific discussion has been updated until 1 May 2004. For information on changes after this date please refer to module 8. 1. Introduction Multiple myeloma Multiple myeloma is a B-cell malignancy of the plasma cell and represents the second most common hematological malignancy, with non-Hodgkin’s lymphoma being the most common. The incidence and prevalence of multiple myeloma are similar in the US and Europe. In 2000, there were approximately 19,000 cases diagnosed in the European Economic Area (Globocan, 2000), with a 5- year prevalence of approximately 47,000 cases. The course of multiple myeloma is characterized by an asymptomatic or subclinical phase before diagnosis (possibly for several years), a chronic phase lasting several years, and an aggressive terminal phase. Multiple myeloma leads to progressive morbidity and eventual mortality by lowering resistance to infection and causing significant skeletal destruction (with bone pain, pathological fractures, and hypercalcemia), anemia, renal failure, and, less commonly, neurological complications and hyperviscosity. From the time of diagnosis, the survival without treatment is between 6 to 12 months and extends to 3 years with chemotherapy. Approximately 25% of patients survive 5 years or longer, with fewer than 5% surviving longer than 10 years. Approved anticancer agents for the treatment of myeloma in the US include melphalan (1992), carmustine (BCNU, 1977) and cyclophosphamide (1959) and in addition, in Europe, epirubicin is also approved for treatment of this disease. At the time of diagnosis, multiple myeloma is a heterogeneous disease, with a course that varies on the basis of both disease- and host-related factors (e.g., age, renal function, stage, alpha2- microglobulin, chromosomal abnormalities).