1

That Nourishes & Nurtures

womanhood

The Feminine Hormone

Estrogen

But Estrogen is an ambivalent steroid hormone, erratic,

inconsistent & mercurial in behavior.

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

Bone Increased deposits of calcium into bone

Increased bone density Osteoporosis

Brain Blocks 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 clots No change in clotting

Blood Fats Increased HDL, decreased LDL, decreased Cholesterol,

Decreased HDL, increased LDL, increased Cholesterol

Skin Increased fat deposits in skin Softer skin Thinner skin, liver spots, dry skin

Uterus Increased 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

3

Molecular Action of Estrogen

Adopted from George et al

hsp90 – heat shock protein90

4

Molecular Action of Estrogen

Adopted from Stanley J Birge et al

AP I – activator proteinCRP – co regulator proteinER – estrogen receptorERE – estrogen response elementPoly II – polymerase IITATA- adenine-thymine-rich sequence important for gene transcription

5

Estrogen Receptor

Two types have been so far identified : - and

Molecular Action of Estrogen

Illustration by Anne Erickson

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

7

Molecular Action of Estrogen

homodimer homodimer & heterodimer• Non-genomic effects

Adopted from George GJM Kuiper et al

Alternating estrogen signaling pathways

8

Molecular Action of EstrogenDifferent response in different

tissues

Adopted from Lewis J. Kleinsmith Ph.D, Donna Kerrigan M.S., Jeanne Kelly

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

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

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

12

Mechanism of Tissue Response - Summary

Oestrogen Receptor Ligand Complex

Oestrogen Receptor

LigandE / SERM / PE/ERD

DNA Oestrogen Response element

Gene Transcriptio

n

Tissue Response

Coregulatory Proteins /

Agonistic & or Antagonistic

AF 1 & 2

Estrogen Signaling in Breast Cancer

In Progress

Tabular Format

Interactive Pathway

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 tissuesERDR

Phytoestrogens

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

16

• Mer 25 (1958)• Clomiphene1. Tamoxifen

• Toremifene• Droloxifene• Iodoxifene

2. Raloxifene3.Ormeloxifene

As of TodaySelective Ostrogen Receptor

Modulators

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

18

The Ideal Selective Ostrogen Receptor Modulator

The perfect SERM

TISSUEEndometriumBreastVaginaBoneLiver/CVSCNS

Perfect AEAEEEEEE-Estrogenic, AE-Anti

Estrogenic

Tamo E

AEAEEE

AE

Ralo AEAEAEE

E+E?

Ormelo

AE AE E E E E

The Search goes on

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 +.

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.

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.

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

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

24

The perfect SERMORMELOXIFENE• 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

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An optimally designed potent SERM with Varied Tissue ResponseOestrogen Antagonist in UTERUS &

BREASTMild Oestrogenic action on Vagina, Bone mineral density, CNS and Serum LipidsNo action on Hypothalamic Pituitary Ovarian function, Thyroid, Adrenal.

The perfect SERMORMELOXIFENE

No Progestational, Androgenic or Antiandrogenic properties

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

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

28

ORMELOXIFENE

Has an excellent safety profile,very well tolerated &

practically without any undesirable side effectsEasy to administer - 60mg tablet

twice a week ( Sunday & Wednesday) for 12 weeks followed

by one tablet of 60mg once weekly

The perfect SERM

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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

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

31

THANK THANK YOUYOU

SERMsWomen have reason to say

SERMs have the potential of providing a new paradigm for

maintaining the health of women.

DNA pol a

Cyclins E,A

B-Myb

Estrogen Receptor

Estrogen Receptors

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

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.

Regulation of ER activity by coactivators and corepressors

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

ER effects on different cell types

Estrogens can activate growth factor receptor signaling

Levin ER. Mol.Endocrinol. 2003;17:309-17

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

Estrogen has multiple effects

Phytoestrogens

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

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

Comparison of binding affinities and transactivation of estrogen and phytoestrogens

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

Dietary Sources of Phytoestrogens

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 0.5-50M which are similar to levels in urine.

Soy Phytoestrogens• Genistein, daidzein, coumesterol, and equol bind to and

transactivate both ER and (0.1-10M)• 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.

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

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.

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.

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%.

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) .

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.

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)

• 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

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 G1 checkpoint seems to be the most important one

LE 12-14G1 checkpoint

G1

S

M

M checkpointG2 checkpoint

G2

Controlsystem

LE 12-15

G1

G1 checkpoint

G1

G0

If a cell receives a go-ahead signal at the G1 checkpoint, the cell continues on in the cell cycle.

If a cell does not receive a go-ahead signal at the G1 checkpoint, the cell exits the cell cycle and goes into G0, a nondividing state.

LE 12-16b

Degradedcyclin G2

checkpoint

S

M

G 2G 1

Cdk

Cyclin isdegraded

MPF Cyclin

Cdk

Molecular mechanisms that help regulate the cell cycle

accumulation

Cyclin

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

LE 12-17

Petriplate

Scalpels

Without PDGF

With PDGF

Without PDGF

With PDGF

10 mm

• 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

LE 12-18aCells anchor to dish surface anddivide (anchorage dependence).

When cells have formed a completesingle layer, they stop dividing(density-dependent inhibition).

If some cells are scraped away, theremaining cells divide to fill the gap andthen stop (density-dependent inhibition).

25 µmNormal mammalian cells

• Cancer cells exhibit neither density-dependent inhibition nor anchorage dependence

LE 12-18b

Cancer cells do not exhibitanchorage dependenceor density-dependent inhibition.

Cancer cells25 µm

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