GnRH analogues

Question 1

Continuous mode of GnRH action

Answer 1

Continuous low-dose/OR single high-dose = Shutting down the axis:
Downregulation of gonadotrophin secretion
Used when gonadal inhibition required i.e. ‘selective medical hypophysectomy’
Equivalent of removing the hypothalamus
NOTE: single high dose will shutdown the axis as there is so much GnRH it remains in the circulation so remains bound to the receptor, so same effect as a continuous low dose.

Question 2

Pulsatile mode of GnRH action

Answer 2

Switching on:
Upregulation of gonadotrophin secretion
Used when stimulation of gonads is required

Question 3

Rationale for native GnRH

Answer 3

Native GnRH
Binds to GnRHR
This stimulates a cellular response -> cellular pathways that results in secrfetion of LH and FSH.

Question 4

Rationale for GnRH analogues: agonists

Answer 4

Bind to GnRHR
Initially triggers same response as native GnRH
HOWEVER this response is only short lived as after a while the response is then terminated and there will be no downstream signalling producing FSH and LH.
SO… the Agonist binds to the GnRH receptor, has same effects as GnRH initially, GnRH has a short half life, GnRH agonist has a longer half life, so the agonist stays there causing a desensitisation causing shutdown of the axis. Agonist does not dissociate from the receptor.
The agonist is initially an activator.

Question 5

Rationale for GnRH analogues: antagonists

Answer 5

Binds to and blocks the GnRHR
There are no downstream events

Question 6

Why do we need GnRH analogues if we are able to synthesise GnRH?

Answer 6

As GnRH has a very short half life (t1/2), so it is degraded shortly after its release.
If administering exogenously we need something with a longer half life
So we need analogues to increase potency & duration of GnRH → analogues created ⇒ agonists or antagonists

They are used to manipulate the HPG axis in clinical practice Eg: IVF, Hormone responsive cancers, endometriosis

Question 6

Native GnRH

Answer 6

Native GnRH is a decapeptide with an amide group attached to a glycine at position 10.
This is highly conserved.

Synthetic GnRH- same primary sequence as endogenous GnRH
Pulsatile mode of delivery = Switching on

Question 7

GnRH structure manipulation

Answer 7

GnRH is a decapeptide with an amide group attached to a glycine at position 10.
This is highly conserved.

Positions 1-4 and 9+10 are highly conserved, these are important residues for GnRHR binding and activation. Amino acid substitutions rarely occur in these areas.

When GnRH has been synthesised and protein folding has occurred, it usually has a horse shoe formation:

Question 8

Positions 1-3 are crucial for…

Answer 8

receptor binding and activation

Question 9

Positions 8-10 are crucial for

Answer 9

receptor binding

Question 10

Position 6, where you have glycine is crucial for

Answer 10

the stability and activation of GnRH

Question 11

what is a d amino acid?

Answer 11

A d amino acid is a stereoisomer of the L amino acid

Answer 12

Position 8, where you have arginine, is where it is most variable across species, where amino acid substitutions occur.

Answer 13

The manipulations and changes being made to create analogues:
D amino-acid substitutions are being made in the regions crucial for receptor binding and activation. They also replace the glycine in position 6 to enhance the stability.

Question 14

GnRH agonists

Answer 14

Making agonists is straightforward
Substitution of Gly (pos 6) with D-amino acids
All agonists & antagonists have substitution of Gly with D-aa at position 6 ⇒ stabilises conformation & enhances activity

In some cases Replacement of Gly-NH2 by NH2-ethylamide binding to Pro (pos 9/10
Replacement of glycine amide with ethylamide at pos 10 to enhance affinity for receptor

Question 15

Making GnRH Agonist brands

Answer 15

Lupron – At position 6 replaced glycine with a d-leucin and replaced the glycine amide with ethylamide. These two changes result in a 10 fold increase in GnRH activity
Buserelin (most popular agonist in IVF) – replaced glycine woth D serine and ethylamide at pos 10. Results in 100 fold increase
Aim is to shutdown he HPG axis so they can take control and start to administer FSH to stimulate follicle growth.

An added benefit in the manipulation of the GnRH structure is that it helps to avoid proteolytic cleavage

Question 16

Making GnRH antagonists

Answer 16

Are a lot more difficult to make. Took 30 years:
1st generation replaced His & Trp at pos 2 & 3 woth d amino acid substitutions, but there was low suppressive activity of the HPG axis.
2nd generation potency increased by D-aa substitution in pos 6 but was withdrawn due to anaphylaxis by histamine release
3rd generation replaced D-Arg by D-ureidoalkayl aa
Usually in position 1-4 and 5-10. So there is a lot more manipulation.

In all the brands, positions 1-3 have been swapped. There is a change in postion 6. Some have a change in position 8. ALL have changes in position 10. There is more manipulation that occurs.
This maintains high binding affinity, blocking GnRHR activation

Question 17

MoA of native GnRH

Answer 17

Binds to the GnRHR
There is an activation of downstream signalling
There is a stimulation of FSH and LH synthesis and secretion
There is dissociation of GnRH from the GnRHR
GnRHR is now responsive to the next GnRH pulse
This is due to the short half life of native GnRH

Question 18

MoA of GnRH agonist

Answer 18

Also binds to the GnRHR
There is the same activation of downstream signalling
There is a stimulation of FSH and LH synthesis and secretion
BUT then there is desensitisation of the GnRHR
GnRHR is resistant to classical desensitisation as there is no c terminal tail (c-terminal tail being phosphorylated and receptor being internalised). In this case, when the agonist remains on the GnRHR, the Gs and Gq pathways get uncoupled from the receptor. (It’s the GnRHR that uncouples from the Gs and Gq pathways).
Now the GnRHR is non-responsive to GnRH

Question 19

MoA of GnRH antagonist

Answer 19

Binds to the GnRHR
Causes blockage of the receptor
There are no downstream effects

Question 20

Clinical use of native GnRH

Answer 20

used for Diagnostic tests (Hypogonadism defined as impaired gonadal function with resultant decreased sex steroids
Used to distinguish between 1° & 2° hypogonadism
Take blood tests and assay GnRH levels
)

Question 21

primary hypogonadism

Answer 21

PRIMARY hypogonadism arises from gonadal failure
As there is no gonadosteroid feedback, there will be an increase in FSH and LH production.

Question 22

secondary hypogonadism

Answer 22

2∘hypogonadism arises from abnormalities of hypo-pituitary axis
Arises from pituitary of hypothalamic abornmalities
So the gonads are functioning but as there is no function of the hypothalamus and pituitary there is NO upregulation of FSH and LH

Question 23

test for hypogonadism

Answer 23

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 (normalish) FSH and LH
Secondary hypogonadism = LOW FSH and LH

Question 24

So if a person has a natural delay in puberty, will GnRH be useful?

Answer 24

GnRH will not be useful as you will not see any response as the pituitary gland of a prepubescent individual is non responsive to GnRH.

(It can be difficult to distinguish between pubertal disorders and constitutive delays in puberty)

Question 25

Native GnRH Hypogonadotrophic hypogonadism (HH)

Answer 25

to diagnose and treat (especially in the case of secondary)
To treat secondary hypogonadism they attach a pump to deliver GnRH in pulses to activate the axis.

Question 26

delayed puberty vs HH

Answer 26

Delayed puberty (there are challenges)
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
Difficult to distinguish between delayed puberty & HH ⇒ pre-pubertal pituitary is unresponsive

Question 27

Clinical uses of GnRH analogues

Answer 27

IVF – most common use
Dysfunctional uterine bleeding
Precocious puberty
Hormone-dependent cancers
Breast cancer
Prostate cancer
Hirsutism and virilisation
Endometriosis

Question 28

Normal HPG axis

Answer 28

In a normal HPG axis there is pulsatile GnRH produced by the hypothalamus onto the anterior pituitary, the is then pulsatile expression of FSH and LH.
As a result, in mid cycle there is selection of the dominant follicle which is followed by ovulation. Then the gonadal steroids will feedback negatively.

Question 29

IVF HPG axis

Answer 29

When we apply the GnRH agonists there is a shutdown of the HPG axis so the menstrual cycle, dominant follicle selection is shutdown. You are essentially uncoupling the HPG axis. Normally agonists are used.
Now you have created a state of pseudo menopause as you have shutdown ovarian function and the HPG axis .
This allows the IFV clinic to administer exogenous FSH to the ovaries to allows the recruitment of multiple follicles (as opposed to just the one).
You need to shutdown the HPG axis before doing this as if you don’t do this and administer exogenous FSH, there is as risk of all of the follicles being ovulated (and therefore lost) also there would be negative feedback that would counteract the process.
Once clinicians have tracked the follicles they trigger ovulation and the final maturation process by administering hCG. It normally is LH in the natural cycle (LH surge) but hCG is used as it has a longer half life than LH and more importantly hCG has LH like properties and can therefore bind and activate the LH receptor. It is more stable in the synthetic for than LH.

Question 30

How does IVF work?

Answer 30

After the HPG axis has been shut down with analogues, and you have administered daily doses of FSH.
Once they can track at least 3 follicles the patient is scheduled for a collection.
They administer hCG to trigger completion of maturation.
36h later they perform the oocyte retrieval using an ultrasound probe connected to a needle.
The oocytes are taken out of the follicular fluid and transferred into a culture. You expect oocytes to be in metaphase 2 stage
Then the sperm is mixed with the oocyte in the culture dish and left to fertilise. Or the sperm can be injected into the egg.
The embryos are cultured up until day 5 – the blastocyst stage where they are transferred back
Embryos are loaded in a catheter with a culture medium, via ultrasound guidance it is inserted into the uterus and expelled.

Question 31

GnRH agonists & IVF

Answer 31

GnRH agonist + gonadotrophins used extensively for follicle growth stimulation in IVF

Major benefit:
improved follicular recruitment ⇒ larger no. oocytes recovered (not in all patients) – as it is very expensive
prevent premature LH surge ⇒ lower cancellation rate
Improvement in routine organisation – as you are in control of hPG axis

Question 32

GnRH agonists & Breast Cancer

Answer 32

Premenopausal women → chemical castration (reduce oestrogen output)
In premenopausal woman, the use of GnRH is the equivalent of chemical castration, as you have shutdown the HPG axis and reduced estrogen output as a lot of breast cancers are estrogen dependant.
GnRHR present in breast cancer tissue (50-60%)
There is a direct anti-proliferative effect of GnRHa in BCa cell lines.

Question 33

GnRH agonists & Prostate Cancer

Answer 33

Prostate Cancer (PCa) is 2nd most frequent tumour in men in West

80% of PCa are androgen dependent

GnRH agonist → desensitisation →↓↓ T (chemical castration)
When you apply the agonist, it shuts down the HPG axis, production of FSH and LH and the production of testosterone. As a lot of these cancers are testosterone dependant.

Question 34

“Flare-effect” results ↑T

Answer 34

Which is the initial action of FSH and LH by the agonist, results in upregulation of testosterone. Its like adding fuel to the fire as prostate cancer is testosterone dependant.

Question 35

why should we Co-administer with anti-androgens?

Answer 35

Micro-surges of T, LH & FSH with continued use

Therefore you Co-administer with anti-androgens

Question 36

GnRH agonists & fertility preservation – female cancers

Answer 36

Last 20 years survival rates of cancers for young women > 80-90%
Large percentage develop POF due to follicular damage from chemo
Chemotherapeutic agents directly attack DNA in dividing and dormant germ cells

To preserve fertility either
Cryopreserve embryos or MII oocytes after IVF and before chemotherapy
Cryopreserve ovarian tissue for transplantation later

Administer adjuvant therapy to minimise gonadal damage?
Use GnRH agonists as a fertility preserveator. Administering GnRH before cancer treatment will minimise damage to the ovary

Question 37

Limitations of GnRH agonists

Answer 37

Temporary solution - symptoms can return

Side-effects -pseudo-menopause in women (with associated symptoms):
reduced libido, erectile dysfunction, increased LDL / decreased HDL cholesterol, insomnia, headaches

Extra pituitary sites of action? (e.g. oocyte, embryo, uterus) in animals - humans??
GnRHR present on these sites – role in implantation? Inadvertently administered during pregnancy

“Flare effect” – Initial upregulation of steroids.

Chronic treatment (>6 months)
Osteoporosis, Heart disease

Question 38

GnRH antagonists & Cancer – (Still under research
Prostate Cancer)

Answer 38

No “flare” or micro-surges. – Advantage
Reduces testosterone to castrate levels by day 3. – Fast acting
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 39

why use hCG instead of LH

Answer 39

• longer half-life
• same effects
• same receptor
• easier to administer?

Question 40

GnRH antagonists advantages

Answer 40

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

Question 41

GnRH antagonists disadvantages

Answer 41

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

Question 42

MoA of GnRH agonist

Answer 42

Binds to GnRH receptor
Initially causes normal activation of signalling that stimulates FSH and LH
Continues to stay bound causing desinsitisation of the GnRHR. Gs and Gq pathways become uncoupled, so it stops responding to GnRH.

Question 43

antagonists are used to..

Answer 43

Antagonist used as:
To prevent flare effect

Question 44

oocyte retrieval vs embryo transfer

Question 45

why do we shut down the HPG axis during IVF?

Question 46

GnRH agonists & fertility preservation – female cancers

Answer 46

• Last 20 years survival rates for young women > 80-90%
• Large percentage develop POF due to follicular damage
• Chemotherapeutic agents directly attack DNA in dividing and
  dormant germ cells
• To preserve fertility either
• Cryopreserve embryos or MII oocytes after IVF and before
  chemotherapy
• Cryopreserve ovarian tissue for transplantation later
• Administer adjuvant therapy to minimise gonadal damage?