GnRH analogues

Question 1

What does continuous low dose/single high dose of GnRH do?

Answer 1

Shuts down HPG axis

Question 2

When would we need to shut down the HPG axis?

Answer 2

• When we want to downregulate gonadotrophin secretion

- When gonadal inhibition is required i.e. ‘selective medical hypophysectomy’

Question 3

When would we need to switch on the HPG axis?

Answer 3

• When we want to upregulate gonadotrophin secretion

- When stimulation of gonads required

Question 4

How do native GnRH/synthetic GnRH act?

Answer 4

binds to receptor, stimulates cellular response and signalling pathways (it’s similar to endogenous GnRH)

Question 5

How do GnRH agonists act?

Answer 5

Mimics pulsatile GnRH functions: switching on HPG axis, same response as native GnRH but after a while it’s shut down because it just stays there and the receptor becomes desensitized, no downstream response

Question 6

How do GnRH antagonists act?

Answer 6

Blocks the receptor by binding to it - Inhibition of GnRH functions and shutting down of HPG axis

Question 7

What is the structure of native/synthetic GnRH?

Answer 7

• same primary sequence as endogenous GnRH
• Decapeptide
• Pulsatile mode of delivery&raquo_space; switches on the HPG axis

Question 8

Why are GnRH analogues used?

Answer 8

GnRH t1/2 in circulation is 2-4 mins so:

• To increase potency & duration of GnRH → analogues created ⇒ agonists or antagonists
• To manipulate the HPG axis in clinical practice - IVF, Hormone responsive cancers, endometriosis

Question 9

Which amino acids of the GnRH are highly conserved?

Answer 9

Amino acids: 1-4 and 9-10

They’re important for GnRHr binding and activation

Question 10

What shape does the GnRH take after synthesis and folding?

Answer 10

horse-shoe configuration

Question 11

Which amino acids are important for receptor binding AND activation

Answer 11

Amino acids 1-3

Question 12

Which amino acids are important for receptor binding ONLY?

Answer 12

Amino acids 8-10

Question 13

Which amino acid is the most variable across species?

Answer 13

Amino acid 8

Question 14

Which amino acids are usually substituted in GnRH antagonists?

Answer 14

Amino acids 1-3 are usually substituted by D amino acids

Question 15

How are GnRH agonists made?

Answer 15

• Substitution of 6 Gly by D-amino acids

* Replacement of Gly-NH2 by NH2-ethylamide binding to Pro (pos 9/10) to enhance affinity for receptor

Question 16

Which amino acid is substituted in agonists and antagonists and why?

Answer 16

• 6 Gly is substituted in all agonists and antagonists by D-aa’s
• The substitutions help avoid proteolytic cleavage
• This stabilises conformation and enhances activity

Question 17

What substitutions happened in the different generation of antagonists?

Answer 17

• 1st generation replaced His & Trp at pos 2 & 3, but low suppressive activity
• 2nd generation potency increased by D-aa substitution in pos 6 but anaphylaxis due to histamine release
• 3rd generation replaced D-Arg by D-ureidoalkayl aa

Question 18

Where do all antagonists have substitutions?

Answer 18

• all antagonists have substitutions in pos 1-3 and pos 10

- There’s more substitutions compared to agonists

Question 19

What is the mechanism of GnRH/synthetic GnRH?

Answer 19

1. Binds to receptor
2. Activation of signalling
3. Stimulation of gonadotropin synthesis and secretion
4. Dissociation of GnRH from GnRHR
5. GnRHR will be responsive to the next GnRH pulse

Question 20

What is the mechanism of GnRH agonists?

Answer 20

1. Binding to the receptor
2. Activation of signalling
3. Stimulation of the gonadotropin synthesis and secretion
4. The agonist remains on the receptor, the Gs and Gq pathways become uncoupled from GnRHR – desensitisation occurs (NOT classical desensitisation)
5. GnRHR non-responsive to GnRH

Question 21

What is the mechanism of GnRH antagonists?

Answer 21

1. Binding to receptor
2. Blockage of receptor
3. No downstream effects

Question 22

What are the clinical uses of native GnRH?

Answer 22

• In the case of gender reassignments and sex change, analogues are used to shut down the HPG axis so other hormones cab be administered
• Diagnostic tests
• To treat hypogonadotropic hypogonadism
• To

Question 23

How is native GnRH used in diagnostic tests?

Answer 23

• To distinguish between 1° & 2° hypogonadism
• Blood test is taken, and GnRH levels are assayed

Test: GnRH is administered intravenously or subcutaneously and plasma LH and FSH are measured at 0, 15, 30, 45 and 60 minutes.
• Primary hypogonadism: High levels of LH/FSH
• Secondary hypogonadism: Low levels of LH/FSH

Question 24

What is the definition of hypogonadism?

Answer 24

Hypogonadism defined as impaired gonadal function with resultant decreased sex steroids

Question 25

What is the difference between primary and secondary hypogonadism?

Answer 25

- 1∘ hypogonadism arises from gonadal failure – no gonado-stereo feedback so there’s a LH/FSH increase - 2∘ hypogonadism arises from abnormalities of hypo-pituitary axis: Gonads are functioning, but the hypothalamus and pituitary doesn’t work properly, downregulation of LH/FSH

Question 26

How is native GnRH used to treat hypogonadotropic hypogonadism?

Answer 26

attach a pump to the individual, GnRH is administered in pulses to activate the HPG axis

Question 27

How is native GnRH used to diagnose delayed puberty?

Answer 27

- Difficult to distinguish between delayed puberty & HH ⇒ pre-pubertal pituitary is unresponsive to GnRH

Question 28

Symptoms of delayed puberty in boys and girls?

Answer 28

* Boys, when testicular growth (volume >4 ml) has not started at 14yrs * Girls, when breast development is not present at 13yrs or menarche did not occur 15-18 years of age

Question 29

What are the common clinical uses of GnRH analogues?

Answer 29

• IVF – most common use • To treat hormone-dependent cancers o Breast cancer o Prostate cancer

Question 30

What are other clinical uses of GnRH analogues?

Answer 30

* Dysfunctional uterine bleeding * Precocious puberty * Hirsutism and virilisation * Endometriosis

Question 31

How is the HPG axis manipulated in IVF?

Answer 31

- continuous GnRH agonist release, the HPG axis is shut down, dominant follicle isn’t selected. - Allows the IVF clinic to add exogenous FSH for the ovaries, multiple follicles recruited instead of one.

Question 32

How is the growth of follicles tracked in IVF?

Answer 32

- Ultrasound is used to track the growth, amount and size of the follicles. - Once follicles have reached required size, ovulation is triggered by administering hCG (it has a longer half-life, and it can bind to the LH receptor)

Question 33

What happens if you don't shut down the HPG axis in IVF?

Answer 33

If you don’t shut down HPG axis: * all of the follicles might get ovulated and then they’re lost * There’s also feedback that counteracts what you’re trying to do

Question 34

When are oocytes in IVF retrieved?

Answer 34

After 36 hours of final maturation, oocytes are retrieved – ovulation happens if you’re too late. Ultrasound probe and a needle is used for this. Needle is used to puncture the follicles and the fluid in the follicles is aspirated. Oocytes also get taken out with the fluid.

Question 35

What happens after oocyte retrieval?

Answer 35

IVF takes place – sperm added to the oocytes. They then looked out for fertilisation. After a few days, embryos are loaded in a capital with a drop of culture media. Via ultrasound guidance it’s inserted into the uterus and expelled from the capital

Question 36

What is used for follicle growth in stimulation and why?

Answer 36

GnRH agonist + gonadotrophins used extensively for follicle growth stimulation in IVF There's major benefit: • improved follicular recruitment >>> larger no. oocytes recovered (not in all patients) • prevent premature LH surge >>> lower cancellation rate • Improvement in routine organisation

Question 37

How are GnRH agonists used to treat breast cancer?

Answer 37

* Premenopausal women → GnRH agonists are used for chemical castration (reduce oestrogen output) * GnRHR present in breast cancer tissue (50-60%) - direct anti-proliferative effect of GnRHa in BCa cell lines

Question 38

How are GnRH agonists used to treat prostrate cancers?

Answer 38

* 80% of prostate cancer (PCa) are androgen dependent * GnRH agonist → desensitisation →↓↓ T (chemical castration) * “Flare-effect” (initial activation of LH/FSH by the agonist) results ↑T >> can be harmful * Micro-surges of T, LH & FSH with continued use * Co-administer with anti-androgens (usually androgen receptor antagonists) – they prevent defects of testosterone

Question 39

Why are GnRH agonists given to female cancer patients?

Answer 39

• Large percentage develop POF (premature ovarian failure) in cancer patients due to follicular damage o Chemotherapeutic agents directly attack DNA in dividing and dormant germ cells * Use GnRH agonists before chemotherapy as a fertility preservative to minimise damage * By shutting down follicular activity it makes the follicles resistant to the damages - but this doesn't guarantee fertility

Question 40

What are the limitations of GnRH agonists in cancer treatment?

Answer 40

* Temporary solution - symptoms can return in the case of cancers * Side-effects: pseudo-menopause in women, reduced libido, erectile dysfunction, increased LDL / decreased HDL cholesterol, insomnia, headaches * Extra pituitary sites of GnRH receptors in animals – might affect these other sites too, we’re not sure how * “Flare effect” with prostate cancers ``` • It's a chronic treatment (>6 months) o Osteoporosis (due to lack of oestrogen), Heart disease (due to changes in the serum lipids) ```

Question 41

Why are GnRH antagonists used to treat prostrate cancers?

Answer 41

* No “flare” or micro-surges. * Reduces testosterone to castrate levels by day 3. * 1st antagonist Abarelix withdrawn due to systemic allergic reaction. * Degarelix >> rapid & sustained reduction in Testo & PSA (prostate specific antigen) routinely used now in advanced prostate cancer.

Question 42

What are the advantages of GnRH antagonists in cancer treatment?

Answer 42

• Rapid action (= rapid pain relief) – 4-6hrs post administered. • Rapid reversal of symptoms • Shorter treatment regime compared to 7-10 days for pituitary down-regulation with agonists. • No “flare effect”. • Dose-dependent. o Can get partial pituitary-gonadal inhibition. o Can adjust level of hypogonadism as desired.

Question 43

What are the disadvantages of GnRH antagonists in cancer treatment?

Answer 43

* Limited licenses available for wider use – only used for specific cases * More expensive than agonists. * Need higher dose than agonist 100mg/month versus 3-5mg. * Competitive inhibitor, therefore less effective over time.