1. Dr S A Ebrahimi

2. Historical overview  1950-1970  Most agents were discovered through large scale screening of natural or synthetic chemicals on rapidly proliferating animal tumors e.g. murine leukemias  Most interacted with DNA synthesis  Paclitaxel and etoposide are examples

3. Last thirty years  Novel molecular targets have been discovered  Some agents are specific for antigens present on some malignant cells e.g. herceptin  Some induce differentiation in malignant cells e.g. all-trans-retinoic acid

4. Current classes of antineoplastic agents  Alkylating agents  Antimetabolites  Natural Products  Hormones and antagonists  Miscellaneous agents

5. Current trends in chemotherapy  Development of molecularly targeted drugs  Use of antibodies growing e.g. herceptin  Use of chemosensitivty testing on biopsied samples prior to drug administration  Use of drug combinations:  Drugs should act via different mechanisms  Each drug used at its highest tolerated dose  Each drug must be administered as frequently as possible  Treatment cycle must be repeated many times to ensure complete eradication of tumor

6. The cell cycle  G1 Phase: Gap period between mitotic phase and DNA synthesis phase  Cells  increase in size  produce RNA  synthesize protein

7. The cell cycle  S phase:  DNA synthesis phase and replication occurs

8. The cell cycle  G2 phase:  Cells continue to produce protein and grow

9. The cell cycle  M phase (mitotic phase):  Cell growth stops  Cell division occurs

10. The cell cycle  Each of the two daughter cells produced in in M phase, can enter G1 phase.

11. The cell cycle  Sometimes, daughter cells enter a non-proliferative state called G0 In slow growing cancers, G0 period is very long

13. The cell cycle and anti- neoplastic agents  Many of the most potent agents damage the DNA and are therefore effective during the S phase  Some other agents block formation of mitotic spindle and are therefore effective during the M phase  Therefore currently, human neoplasms which are susceptible to chemotherapy are those with a large proportion of cells undergoing division  Also normal cells with high rate of division e.g. bone marrow and hair follicles, are also affected by these agents  Cancers with low percentage of dividing cells e.g. carcinoma of colon, show little susceptibility to these agents

14. The cell cycle  Each transition is controlled by:  Cyclins: Kinase activating proteins  Cyclin-dependent kinases (CDK)  Inhibitory proteins e.g. p16 and retinoblastoma protein  Loss of Inhibitory proteins or enhanced activity of CDKs cause excessive proliferation  Research is currently focused on CDK’s as drug targets

15. The cell cycle  At phase boundaries, check points exist for examining DNA integrity  G1-S boundary: If the protein p53 is expressed normally  cells are checked for DNA damage in G1  cells with damaged DNA undergo apoptosis  G2-M boundary:  DNA integrity is checked again Mutation in check point components can induce drug resistance to cancer cells

16. Achieving optimal efficacy from chemotherapy  Treatment of cancer is the application of a complex mixture of  Radiotherapy  Surgery  Chemotherapy  “standard” chemotherapy regimens have been devised for different cancers  Efficacy not optimal in all patients  Side-effects are usually great

17. Achieving optimal efficacy from chemotherapy  In order to limit side-effects and optimize efficiency  Individual dose adjustments are made based on:  Body surface area but No solid data supports this use  Recently  Pharmacokinetic monitoring has been shown to:  Increase efficiency e.g. methotrexate in ALL treatment but maintaining a targeted plasma level  Decreasing toxicity e.g. thrombocytopenia induced by carboplatin by dose adjustment based on renal clearance

18. Achieving optimal efficacy from chemotherapy  Selecting the right treatment for a particular patient  Finding therapy responders  Checking existence of CD20 antigen prior to treatment with rituximab  Testing for HER2 receptor existence prior to treatment with trastuzumab antibody  Chemosensitivity testing  Checking drug metabolizing enzyme polymorphisms  Risk of toxicity in patients with polymorphisms of dihydropyrimidine dehydrogenase gene on 5-fluoruracil treatment  Breast cancer resistance gene profiling prior to treatment

19. Management of toxicity  Antineoplastic agents have variable kinetics and toxicity  Have to monitor for:  Blood cell count  Infections  Delayed toxicities on the:  Heart  Kidneys  Lungs

20. Current classes of antineoplastic agents  Alkylating agents  Antimetabolites  Natural Products  Hormones and antagonists  Miscellaneous agents

21. Alkylating agents  Goodman and Gilman discovered cytotoxic effects of mustard gas on murine lymphomas  Subsequently tested a number of agents clinically  Five major groups are used today:  Nitrogen mustards  Ethyleneimines  Alkyl sulfonates  Nitrosoureas  Triazenes

22. Alkylating agents  They all produce a carbonium intermediate  Carbonium can attack a nucleophile such as  Phosphate  Amino  Sulfhydryl  Hydroxyl  Carboxyl  Imidazole

23. Alkylating agents  Nitrogen number 7 (N7) on guanine is particularly sensitive to this alkylation  Guanine is probably most important biological target  Other atoms in DNA susceptible to attack are:  N1 and N3 of adenine  N3 of cytosine  O6 of guanine  The result is cross linking of DNA chains to each other or to proteins

24. Pharmacological actions  Inhibition of DNA synthesis  Inhibition of cell division  Rapidly dividing cells are affected more  Delayed damage also seen in tissues with low mitotic indices:  liver  kidney  mature lymphocytes

25. Monofunctional versus bifunctional agents  Bifunctional agents  create interstrand cross-links  This stops DNA replication with little chance of DNA repair  Monofunctional agents  alkylate the chains, but no cross-linkages are formed  DNA repair processes may be able to overcome the damage  Mutations may occur because of alkylation-repair sequence  Causing drug resistance  Carcinogenesis is normal tissue

26. Mechanisms of resistance in alkylating agents  Decreased drug transport  Increased intracellular nucleophile concentrations e.g. increased glutathione  Increased activity of DNA repair mechanism  Increased rate of metabolism of active drugs

27. Toxicity of alkylating agents  Dose-dependent bone marrow suppression  Acute myelosuppression  Peak in 6-10 days after initiation of therapy  Recovery in 14-21 days after cessation of therapy  Mucosal toxicity  Oral mucosal ulceration  Intestinal denudation  Neurotoxicity  Nausea and vomitting  Some agents induce seizures, cerebellar ataxia

28. Therapeutic uses of Nitrogen mustards  Mechlorethamine  As part of MOPP regimen (mechlorethamine, vincrsitine (oncovin), procarbzaine and prednisone) for the treatment of Hodgkin’s disease (cancer of the lymph tissue as in lymph nodes, spleen etc)  Topically for the treatment of cutaneous T-cell lymphoma

29. Therapeutic uses of Nitrogen mustards  Cyclophosphamide  Breast cancer  Lymphomas  Chronic lymphocytic leukemia  Non-Hodgkin’s lymphomas  Ovarian cancer  Solid tumors in children  Burkitt’s lymphoma, associated with Epstien- Barr virus, complete remission reported  Also as an immunosuppressant

30. Therapeutic uses of Nitrogen mustards  Ifosfamide  Germ cell testicular cancer  Sarcomas  Melphalan  Multiple myeloma  Chlorambucil  Chronic lymphocytic leukemia (CLL)

31. Other alkylating agents  Altretamine  Persistent or recurrent ovarian cancer when cisplatin or other agents have failed  Busulfan  Chronic myeloid leukemia  Carmustine  It passes the blood-brain barrier  Used in treatment of Malignant gliomas

32. Other alkylating agents  Dacarbazine  Hodgkin’s lymphoma  Less effective for treatment of melanoma’s and adult sarcomas

33. Platinum coordination complexes  Platinum complexes were found to have antiproliferative activity in the 1960’s  Cis-diaminedichloro-platinum (II) was the most potent  It inhibits DNA synthesis by formation of inter and intra strand cross-linkages.  N7 of guanine appears to be most susceptible to the attack

34. Other alkylating agents  Platinum coordination complexes (Cisplatin)  Have broad antineoplastic activity  With etoposide, vinblastine, bleomycin or ifosfamide, cis- platin cures 90% of cases of testicular cancer  In carcinoma of ovaries, with paclitaxel, induces complete response in most cases  Used for the treatment of:  Carcinomas of the lung  Cancers of neck, head, bladder, endometrium and cervix  Rectal and anal carcinomas  Enhances effects of irradiation in some cancers e.g. esophegal and lung

35. Antimetabolites  Folic acid analogs:  Historically important  1 st agents to produce temporary remission in leukemia  1 st agents to produce cure of a solid tumor (choriocarcinoma of the uterus)

36. Structures of folic acid analogs

37. Folic acid: Mode of action  Deoxyuridine monophosphate is converted to thymidine monophosphate using tetrahydorofolate and producing dihydrofolate  The enzyme dihydrofolate reductase, converts dihydrofolate to tetrahydrofolate  The cycle can repeat for the production of the next TMP molecule

38. Folic acid analogs: Mode of action  Main mechanism of action is to inhibit dihydrofolate reductase  Dihydrofolate is not reduced to tetrahydrofolate  Tetrahydrofolate reserves become depleted  Production TMP is inhibited  Production of DNA strands becomes inhibited  Cell division is blocked

39. Methotrexate  Critical in the treatment of acute lymphoblastic leukemia (ALL) in children  Of little value in adult leukemias except leukemic meningitis  With dactinomycin, can produce cure in 75% of advanced cases of choriocarcinoma and 90% of early diagnosed cases  Beneficial effects are seen in combination therapies in Burkitt’s lymphomas  Is a component of drug regimens in treatment of carcinomas of the:  Breast, head, neck, ovary and bladder

40. DNA nucleotides  Purines: Two fused rings  Adenosine  Guanine  Pyrimidines: Single 6 member rings  Thymine  Cytosine  Uracil  The bases are converted to deoxynucleoside triphosphates (dNTP)  dNTP is the substrate for DNA polymerase

41. Pyrimidine analogs  5-fluorouracil  It is transformed to 5-fluoro-2-deoxyuridine-5-phosphate (FdUMP)  FdUMP covalently inhibits the enzyme Thymidylate synthetase reposnsible for synthesis of TMP  Thus DNA synthesis becomes inhibited  It also is incorporated into RNA  Thus interferes with RNA function  Used with some success, for treatment of carcinomas of the colon, upper digestive tract and breast

42. Cytarabine (cytosine-arabinoside)  Enters the cell via active transport mechanism  Becomes incorporated into DNA during synthesis  Inhibits Base stacking and normal DNA conformation  Interferes with DNA replication  Effective in  Acute Myelocytic Leukemia  Newer Agents:  Azacitidine  Gemcitabine

43. Purine analogs  6-Mercaptopurine  Converted to 6-thioinosine-5-monophosphate (T- IMP)  T-IMP accumulation:  Inhibits formation of purine bases  To a small extent is incorporated into DNA  Used for the treatment of acute leukemia

44. Natural products  Vinca alkaloids  Obtained from a plant indigenous to Madagaskar  Three clinically important agents have been isolated  Vincristine  Vinblastine  Vinorelbine

45. Vinca alkaloids: mode of action  These are cell-cycle-specific agents  They bind beta-tubulin stiochiometrically  Alkaloid bound tubulin can not polymerize with alpha-tubulin to form microtubules  Mitotic spindle can not form  Chromosomes can not organize themselves at the mitotic plate  Mitosis does not proceed  Cells undergo apoptosis

46. Vinblastin  Curative in combination with bleomycin and cisplatin for treatment of testicular cancer  A component of curative therapy for Hodgkin’s disease  Acitve in:  Kaposi’s sarcoma due to infection by human herpes virus 8 (HHV8)  Neuroblastoma  Carcinoma of the breast  Choriocarcinoma

47. Other vinca alkaloids  Vincristine  With glucocorticoids as treatment of choice in childhood leukemia  As part of MOPP regimen for treatment of adult lymphomas  Vinorelbine  Non small cell carcinoma of the lung with cisplatin  Carcinoma of the breast

48. Taxanes  Paclitaxel was 1 st isolated from the bark of Yew tree  It binds beta-tubulin at a site different from vincristine  It promotes microtubule formation

49. Taxanes: mode of action  Binds beta-tubulin  Antagonizes break down of microtubules  The cell becomes locked in the mitotic phase  Cell death follows

50. Taxanes: Uses  Paclitaxel  Metastatic cancers of:  ovaries  Breast  Lung  head and neck  Docetaxel  Hormone-refractory prostate cancer

51. Camptothecin  Initially isolated from a Chinese tree in 1966  It was found to be too toxic in vivo for clinical application  Clinically useful analogs were developed in the 1980’s

52. Camptothecin analogs: mode of action  Topoisomerases are enzymes that reduce torsional stress in a selected region of DNA  This is achieved by untangling the DNA strand  This allows the two DNA strands to separate  Separation is necessary before  Replication  Repair  Transcription  Topoisomerase are a family of enzymes with two subtypes: I and II  These agents are inhibitors of Topoisomerase I

53. Camptothecin analogs: mode of action  Tompoisomerase I binds covalently to double stranded DNA through a reversible trans- esterfication reaction  This reaction attaches tyrosine on the enzyme to phosphate on the DNA  This causes a break in the DNA strand to which the enzyme has attached  The other end of the DNA strand is free to rotate, unraveling the DNA  The enzyme then reattaches the two broken ends of DNA

54. Camptothecin analogs: mode of action  Camptothecins binds the topoisomerase I- DNA complex  Stabilises the normally reversible ester bond.  The agents do not affect the rate of formation of topoisomerase-DNA complex  Religation becomes inhibited  Single strand-breaks become accumulated  This makes normal DNA replication impossible  Cells ultimately undergo apoptosis

55. Camptothecin analogs: mode of action  These agents are S-phase specific  Trials have shown that low dose, long term usage is more effective that high dose short term use  There is some cytotoxicity in cells which are not synthesizing DNA  This suggests a mixed effect

56. Clinical use  Topotecan  Ovarian cancer  Small cell lung cancer  CML  Irinotecan  With fluoropyrimidines in advanced colorectal cancer in patients which have not been treated before  Other possible uses include:  Small cell and non-small cell long cancer  Cervical, gastric, ovarian cancers and brain tumors

57. Antibiotics  Dactinomycin (Actinomycin D)  The palanar sing structure appears to intercalate between adjacent guanine-cytosine base pairs  The amino acid chains align themselves along the minor groove  A stable dactinomycin-DNA complex is formed  The complex inhibits DNA and RNA polymerases  Also, agent appears to induce nicks in the DNA structure

58. Dactinomycin  Is cytotoxic to rapidly dividing cells  Is used clinically for  Rhabdomyosarcoma in children  Kaposi’s sarcoma  Soft tissue sarcoma’s  With methotrexate, has been used in advanced choriocarcinoma

59. Anthracyclin antibiotics  A tetracycline ring structure attached to a sugar, daunosamine  A number of mechanisms suggested for their antitumor activity

60. Anthracyclin antibiotics: Mode of action  These agents form a complex with DNA- bound topoisomerase II enzyme  This, inhibits religation of nicked DNA strand  Normal DNA replication and repair become impossible  Cells under apoptosis  Also, the agents generate free radicals  Important in their cardiotoxicity

61. Anthracyclin antibiotics: Uses  Daunorubicin  AML  AIDS-related kaposi’s sarcoma  Doxorubicin  Kaposi’s sarcoma  Malignant lymphomas  Carcinoma of breast  Small cell carcinoma of the lung

62. Epipodophyllotoxins  Extracted from Mandrake tree, endogenous to north America  They form a complex with DNA bound topoisomerase II  This leads to cell death

63. Epipodophyllotoxins  Etoposide  Testitular cancer  Small cell carcinoma of the lung  Non-Hodgkin’s lymphomas  Kaposi’s sarcoma  May cause acute nonlymphocytic leukimia  Teniposide  Glioblastomas  Neuroblastomas  Brain metastases from small cell lung carcinoma

64. Bleomycin  Appears to induce single and double stranded breaks in the DNA  This is through oxidative damage to the deoxyribose of thymidylate  Requires Fe and oxygen for its actions  It causes accumulation of cells in G2 phase  Used for treatment of  Germ cell tumors of testis and ovaries  Malignant pleural effusions  As part of ABVD therapy in Hodgkin’s disease

65. L-Asparginase  An enzyme which converts aspargine to aspartic acid  This decreases free serum aspargine  As cells in some lymphoid malignancies can not synthesize this amino acid, their proliferation becomes inhibited  Used in combination for treatment of acute lymphoblastic leukemia (ALL)

66. Differentiating agents  Tretinoin  A retinoid  Under physiological conditions  Retinoic acid receptor-alpha (RAR-alpha) dimerizes with RAR-X receptor to form a complex with all-trans- retinoid acid (ATRA)  This complex induces cell differentiation i.e. stops malignant proliferation of cells  In some malignancies, the level of ATRA is too small to form the tripartite complex in adequate amounts  Tretinoin is given to compensate low ATRA levels  Very effective for Acute promyelocytic leukemia

67. Tyrosine kinase inhibitors  Human genome contains code for 550 different protein kinases  These can be divided into 3 groups  Tyrosine Kinases  Receptor tyrosine kinases (have extracellular ligand binding site)  Simple Enzymatic (in cytoplasm or nuclear compartment)  Serine/threonine kinases  Nonselective kinases (serin,threonine and tyrosine)

68. Tyrosine kinase inhibitors  The enzyme acts as on-off switch for many protein functions  Some mutations cause the enzyme to remain locked in the “on” position, leading to malignant proliferation  Subtypes of enzyme implicated in cancers include  Platelet derived growth factor receptor  Kit: a growth factor receptor of type III tyrosine kinase family  ABL-Kinase

69. Tyrosine kinase inhibitors  Three agents have obtained FDA approval:  Imatinib  CML (ABL positive)  GIST (Kit Mutation positive)  Gefitinib  Erlotinib

70. Thalidomide  A number of mechanisms have been proposed for the effects  Direct cytotoxic/proapoptotic effects  Inhibition of cytokine production, release and signaling, leading to antiangiogenic effects  Immunostimulatory effects, enhaning natural killer cells cell-mediated cytotoxicity  Used in multiple myeloma treatment

71. Other agents  Interleukin-2  Monoclonal antibodies  Naked  Trastuzumab (herceptin) for the treatment of breast cancer  Conjugated to cytotoxic agents  Gemtuzumab  Treatment of acute myelocytic leukemia

72. Hormones  Glucocorticoids  In acute lymphoblastic leukemia in children  Malignant lymphomas in children  Progestins  Metastatic hormone dependent breast cancer  Anti-androgen therapy  Metastatic prostatic cancer  Anti-estrogen therapy  Tamoxifen for breast cancer

73. Research in our lab  Spinal-Z  Two polymethoxy flavones  Can inhibit cell growth in vitro  Can inhibit tumor growth in vivo

74. Research in our lab  Found them to be anti-angiogenic

75. Summary of mode of action of antineoplastic agents