2. 1 That Nourishes & Nurtures womanhood The Feminine Hormone Estrogen But Estrogen is an ambivalent steroid hormone, erratic, inconsistent & mercurial in behavior.

3. 2 Effects of Estrogen at Various Sites in the Body Tissue Effect of Estrogen Stimulation Clinical Effect of Stimulation Clinical Effect of Absence of Stimulation BoneIncreased deposits of calcium into bone Increased bone density Osteoporosis BrainBlocks the release of ovarian estrogen None Hot flashes, sleep disorders, mood changes, problems with memory? Alzheimer’s disease?? Breast Stimulates growth of breast tissue Bigger breasts,? Increased risk of breast cancer, increased sensitivity of the breast, Smaller breasts Blood Clotting Increased risk of blood clotsNo change in clotting Blood Fats Increased HDL, decreased LDL, decreased Cholesterol, Decreased HDL, increased LDL, increased Cholesterol SkinIncreased fat deposits in skinSofter skinThinner skin, liver spots, dry skin UterusIncreased stimulation of uterine lining and muscle Heavier cycles, increased risk of uterine cancer No periods Vagina Increased thickening of skin, better blood supply to tissue Vaginal discharge, feelings of pelvic congestion Dryness, vaginal infections, painful sex, incontinence of urine, pelvic weakness

4. 3 Molecular Action of Estrogen Adopted from George et al hsp90 – heat shock protein90

5. 4 Molecular Action of Estrogen Adopted from Stanley J Birge et al AP I – activator protein CRP – co regulator protein ER – estrogen receptor ERE – estrogen response element Poly II – polymerase II TATA- adenine- thymine-rich sequence important for gene transcription

6. 5 Estrogen Receptor Two types have been so far identified : -  and  Molecular Action of Estrogen Illustration by Anne Erickson

7. 6 Estrogen Receptor Distribution  &  - CNS, blood vessels, bone, heart, breast, ovary, uterus, testes, prostate  - Liver  - Lungs, kidney, bladder, intestines Adopted from George GJM Kuiper et al *Based on the level of ER mRNA levels *Awaits confirmation till subtype specific monoclonal antibodies are available Molecular Action of Estrogen

8. 7  homodimer  homodimer  &  heterodimer Non-genomic effects Adopted from George GJM Kuiper et al Alternating estrogen signaling pathways

9. 8 Molecular Action of Estrogen Different response in different tissues Adopted from Lewis J. Kleinsmith Ph.D, Donna Kerrigan M.S., Jeanne Kelly

10. 9 Estradiol Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex®), development of a novel, "pure" antiestrogen. Cancer 2000; 89: 819. Molecular Action of

11. 10 SERM(Tamoxifen) Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex ® ), development of a novel, "pure" antiestrogen. Cancer 2000; 89: 819. Molecular Action of

12. 11 Estrogen Receptor Down regulator A Promising Area of Research Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex ® ), development of a novel, "pure" antiestrogen. Cancer 2000; 89: 819. Molecular Action of

13. 12 Mechanism of Tissue Response - Summary Oestrogen Receptor Ligand Complex Oestrogen Receptor Ligand E / SERM / PE/ERD DNA Oestrogen Response element Gene Transcription Tissue Response Coregulatory Proteins  /  Agonistic & or Antagonistic AF 1 & 2

14. Estrogen Signaling in Breast Cancer In Progress Tabular Format Interactive Pathway

15. 14 Selective Ostrogen Receptor Modulators Estrogens Anti Estrogens SERMs SERMs- designed to act in specific ways at each of the oestrogen receptor sites in different tissues ERDR Phytoestrogens

16. 15 Designer drugs which exhibit tissue specific desirable Estrogenic & Antiestrogenic actions in different tissues “Designer Estrogens” “Fantasy Estrogens” They have the potential of providing a new paradigm for maintaining the health of women. Selective Ostrogen Receptor Modulators

17. 16 Mer 25 (1958) Clomiphene 1.Tamoxifen Toremifene Droloxifene Iodoxifene 2.Raloxifene 3.Ormeloxifene As of Today Selective Ostrogen Receptor Modulators

18. 17 The Ideal Selective Ostrogen Receptor Modulator The perfect SERM The ideal SERM is one that prevents bone loss, has no risk of uterine or breast cancer, a +ve effect on lipids & cardiovascular system, relieves PMS and maintains cognitive function of the brain The Search goes on Adopted from – Rita de Cassia M Dardes & V Craig Jordan

19. 18 The Ideal Selective Ostrogen Receptor Modulator The perfect SERM TISSUE Endometrium Breast Vagina Bone Liver/CVS CNS Perfect AE E E-Estrogenic, AE-Anti Estrogenic Tamo E AE E AE Ralo AE E E+ E? Ormelo AE  E  The Search goes on

20. 19 Tamoxifen The first true SERM. In use for breast cancer treatment since 1968, 10m patient use years. Approved for prophylactic use in1997. Beneficial effect on osteoporosis. Effect on CVS +? –Lipid profile +.

21. 20 Tamoxifen Has many undesirable E / AE actions. –E in uterus – risk of End. Cancer. –Alleged as a carcinogen. –AE in vagina, CNS? Unsatisfactory safety/toxicity profile. Gave boost to the continued research for SERMs. Under evaluation-star trial-6/99, 22000 women for 5-10 yrs.

22. 21 Raloxifene Originally approved (1998) for use for treatment and prevention of osteoporosis. Subsequently (1999) approved for breast cancer prevention after ‘MORE’ study Improved safety profile than Tamoxifen Cardiovascular effects are unequivocal & under evaluation.

23. 22 Raloxifene  Risk of venous thromboembolism No effect on endometrium. AE on vagina Effect on CNS?. No improvement in cognitive function Does not relieve PM hot flashes Possible future use as HRT?? Is on evaluation- STAR trial

24. 23 ORMELOXIFENE The individual elements of the molecular structure give a tissue selectivity- different DNA transcriptions in different tissues Estrogen agonist Estrogen antagonist Chemical Name- Trans -7-methyl-2-2- dimethyl-3-phenyl-4(4-(2- pyroldinoethoxy)phenyl(-chroman hydrochloride), related to centchroman The perfect SERM

25. 24 The perfect SERM ORMELOXIFENE Enhanced tissue selectivity –Basic amine side chain – uterine AE action –Pyrolidine base – highest degree of antagonistic action –Benzopyran group – agonistic action & binding affinity Very strong binding affinity to ER –Quick & potent action Slow nuclear build up & prolonged retention of ER –Long half life & prolonged action

26. 25 An optimally designed potent SERM with Varied Tissue Response Oestrogen Antagonist in UTERUS & BREAST Mild Oestrogenic action on Vagina, Bone mineral density, CNS and Serum Lipids No action on Hypothalamic Pituitary Ovarian function, Thyroid, Adrenal. The perfect SERM ORMELOXIFENE No Progestational, Androgenic or Antiandrogenic properties

27. 26 ORMELOXIFENE Special benefit in perimenopausal women – Relief of PMS Currently indicated for the treatment of Dysfunctional Uterine Bleeding at ANY AGE. Not suitable for women desiring pregnancy Approved for inclusion in National Family Welfare Program, for social marketing. The perfect SERM

28. 27 ORMELOXIFENE Contraindicated in – H/O recent liver dysfunction or clinical jaundice PCOD Cervical Dysplasia & Chronic Cervicitis Hypersensitivity to the drug Allergic conditions Nursing mothers Chronic illness The perfect SERM

29. 28 ORMELOXIFENE Has an excellent safety profile,very well tolerated & practically without any undesirable side effects Easy to administer - 60mg tablet twice a week ( Sunday & Wednesday) for 12 weeks followed by one tablet of 60mg once weekly The perfect SERM

30. 29 ORMELOXIFENE  Currently being evaluated for use in the treatment and prevention of: - Breast Cancer Osteoporosis  Possible future use: - Menopause management Fibromyoma Endometriosis and Adenomyosis Contraceptive The perfect SERM

31. 30 ORMELOXIFENE WARNING: -  Indian contribution  Not introduced in the international arena  Not approved by FDA  Not yet fully evaluated - extensive clinical trials needed The perfect SERM

32. 31 THANK YOU THANK YOU SERMs Women have reason to say SERMs have the potential of providing a new paradigm for maintaining the health of women.

35. DNA pol a Cyclins E,A B-Myb

36. Estrogen Receptor

37. Estrogen Receptors http://www.bio.cmu.edu/Courses/BiochemMols/ER/#ERchime

38. Estrogen Receptors ER-  –Uterus, testis, pituitary, ovary, epididymis, and adrenal gland. ER-  (Kuiper et al. 1996) –brain, kidney, prostrate, ovary, lung, bladder, intestine, and epididymis. –88% identity with rat ER-   identity with human ER-  Membrane localized ER (Pietras and Szego, 1997) ER  and  differ in C-terminal ligand binding domains and N-terminal transactivation domains. Highest homology in DNA binding domain.

40. Regulation of ER activity by coactivators and corepressors

42. Hall et al. 2001. J. Biol. Chem., 276: 36869-36872

43. ER effects on different cell types

44. Estrogens can activate growth factor receptor signaling Levin ER. Mol.Endocrinol. 2003;17:309-17

45. Belcher & Zsarnovszky, 2001. J. Pharmacol. Exp. Therap. 299:408-414.

46. Estrogen has multiple effects

49. Phytoestrogens Aherne and O’Brien, 2002. Nutrition 18:75-81.

50. Benassayag, et al., 2002. J. Chromatogr.B 777:233-248.

51. Comparison of binding affinities and transactivation of estrogen and phytoestrogens Belcher & Zsarnovszky, 2001. J. Pharmacol. Exp. Therap. 299:408-414

52. Dietary Sources of Phytoestrogens

53. Pytoestrogens in humans Phytoestrogens have weaker estrogenic activity compared to circulating estrogens (17-  -estradiol or estrone). Phytoestrogens can bind sex steroid binding protein (SBP) and  -feroprotein (AFP) and be circulated. Dietary phytoestrogens are metabolised by intestinal bacteria, absorbed, conjugated in the liver (by sulfotransferases and UDP-glucoronyosyl transferases), circulated in plasma and excreted in urine. Phytoestrogen levels are higher in fluid collected from breast and prostatic ducts compared with serum or plasma. Urinary isoflavonoid excretions range from about 0.3-30  M/day. Urinary secretions of vegetarians may contain 1000 times higher phytoetsrogens than total urinary steroid estrogens. Phytoestrogens demonstrate inhibitory effects at 0.5-50  M which are similar to levels in urine.

54. Soy Phytoestrogens Genistein, daidzein, coumesterol, and equol bind to and transactivate both ER  and  (0.1-10  M) Genistetin has a higher affinity for ER . Soy PEs effect cell cycle progression, growth, and differentiation. Have antioxidant and anti ‑ angiogenic activities. Genistein affects cellular function via inhibition of 17 beta- steroid oxidoreductase (an enzyme necessary for conversion of androgens to E2). Inhibits aromatase. Effects cycloxygenase, lipoxygenase, Cholesterol 7  hydroxylase. Modulates the activity of topoisomerase II. Modulates enzymes involved in phosphoinositide (PI) turnover. Modulates TGF-β signaling cascades Increases epidermal growth factor (EGF) and EGFR levels.

55. Genistein Both estrogenic and anti-estrogenic effects Inhibitor of tyrosine kinases 20-fold higher binding affinity for ER-  than ER-  (Makela et al. 1999) 4',5,7-Trihydroxyisoflavone

56. Phytoestrogens in Human Health Cancer preventive Post-menopausal supplement Prevention of osteoporosis Cardiovascular health Fertility Breast enhancement References: Kurzer, 2003. J. Nutr. 133: 1983S-1986S. Benassayag, et al., 2002. J. Chromatogr.B 777:233-248.

57. Cancer preventive Benefits to human breast and uterine cancer controversial. Genistein can be carcinogenic in uterine cancer at neonatal exposure. Cancer protective in animal studies, especially when exposed during breast development. Isoflavonoids and lignans stimulate proliferation of ER+ breast cancer cells. Inhibit cell growth at high concentrations and in ER  (-) breast cancer cells. Therefore, ER  may have cancer protective effect. Anti-angiogenic effects of genistein, daidzein, and biochanin A may contribute to antitumor activity. Anti-oxidants in vitro and in vivo.

58. Post-menopausal therapy In 2002, the Women’s Health Initiative (WHI) trial of estrogen/progestin therapy was halted midtrial due to high incidence of breast cancer and cardiovascular disease. Consumption of 30mg/d soy isoflavones may reduce hot flashes by 30-50%.

59. Prevention of osteoporosis Isoflavone intake increases bone mineral density. Can be useful in preventing post- menopausal osteoporosis. Diets rich in phytoestrogens can protect long-term bone loss (Setchell & Lydeking-Olsen, 2003. Am. J. Clin. Nutr. 78:593S- 609S).

60. Cardiovascular health Average intake of 47g/day soy protein results in 9% decrease in total cholesterol,13% decrease in LDL cholesterol, and a trend towards HDL cholesterol. Flavanoids decrease platelet aggregation. Genistein-induced inhibition of growth factor activity can interfere with platelet and thrombin action.

61. Effects on fertility (premenopausal) Interferes with menstrual cycle (delay) Reduced LH and FSH and progesterone. Male rodents exposed to PEs in early life: impaired semen quality, congenital malformations, testicular cancer (coumesterol, delay in mating)

62. Antioxidant, anti ‑ apoptosis, anti ‑ inflammatory, anti-cancer, and anti ‑ invasive. Reduces Cu-induced LDL oxidation by binding to LDL via a glycosidic ether bond. Increases HDL cholesterol. Inhibits platelet activation. Ameliorates neuronal damage due to ethanol consumption. Probably via antioxidant effect. Minimizes effects of NOS activity by ehtanol. Inhibits ethanol-induced arachidonic acid release and cycloxygenase activity. Anti-ageing role? inhibitory effects on cancer initiation, growth promotion progression and angiogenesis in model systems. The anti ‑ proliferative activity of resveratrol is mediated by p38-MAPKs via p53 mediated inhibition. Resveratrol may inhibit apoptosis induced by oxidized lipoproteins through inhibition of NF-  B and AP-1 pathways. Resveratrol inhibits protein kinase C, Akt, and FAK activities in ER  (+) breast cancer cells. Red wine phytoestrogens: Resveratrol, quercetin, and anthocyanins

63. The Cell Cycle Control System The sequential events of the cell cycle are directed by a distinct cell cycle control system, which is similar to a clock The clock has specific checkpoints where the cell cycle stops until a go-ahead signal is received For many cells, the G 1 checkpoint seems to be the most important one

64. LE 12-14 G 1 checkpoint G1G1 S M M checkpoint G 2 checkpoint G2G2 Control system

65. LE 12-15 G1G1 G 1 checkpoint G1G1 G0G0 If a cell receives a go-ahead signal at the G 1 checkpoint, the cell continues on in the cell cycle. If a cell does not receive a go-ahead signal at the G 1 checkpoint, the cell exits the cell cycle and goes into G 0, a nondividing state.

66. LE 12-16b Degraded cyclin G 2 checkpoint S M G2G2 G1G1 Cdk Cyclin is degraded MPF Cyclin Cdk Molecular mechanisms that help regulate the cell cycle accumulation Cyclin

67. Stop and Go Signs: Internal and External Signals at the Checkpoints An example of an internal signal is that kinetochores not attached to spindle microtubules send a molecular signal that delays anaphase Some external signals are growth factors, proteins released by certain cells that stimulate other cells to divide For example, platelet-derived growth factor (PDGF) stimulates the division of human fibroblast cells in culture

68. LE 12-17 Petri plate Scalpels Without PDGF With PDGF Without PDGF With PDGF 10 mm

69. Another example of external signals is density-dependent inhibition, in which crowded cells stop dividing Most animal cells also exhibit anchorage dependence, in which they must be attached to a substratum in order to divide

70. LE 12-18a Cells anchor to dish surface and divide (anchorage dependence). When cells have formed a complete single layer, they stop dividing (density-dependent inhibition). If some cells are scraped away, the remaining cells divide to fill the gap and then stop (density-dependent inhibition). 25 µm Normal mammalian cells

71. Cancer cells exhibit neither density- dependent inhibition nor anchorage dependence

72. LE 12-18b Cancer cells do not exhibit anchorage dependence or density-dependent inhibition. Cancer cells 25 µm

73. Loss of Cell Cycle Controls in Cancer Cells Cancer cells do not respond normally to the body’s control mechanisms Cancer cells form tumors, masses of abnormal cells within otherwise normal tissue If abnormal cells remain at the original site, the lump is called a benign tumor Malignant tumors invade surrounding tissues and can metastasize, exporting cancer cells to other parts of the body, where they may form secondary tumors