Lecture 9: GnRH Analogues Flashcards
What are GnRH analogues?
- GnRH agonists
- GnRH antagonists
Clinically, how can we shut down the HPG axis?
- Continuous low-dose/single high-dose =SHUTTING DOWN
What do we see when the HPGF axis is shut down?
- Down-regulation of gonadotrophin (LH/FSH) secretion
When is it useful to shut down the HPG axis?
- When gonadal inhibition is required i.e. ‘selective medical hypophysectomy’ or shutting down ovaries for IVF
Clinically, how can we switch on the HPG axis?
- Pulsatile mode of delivery
What is characterised by the switching on of the HPG axis?
Up-regulation of gonadotrophin secretion
When may we switch the HPG axis on?
- When stimulation of gonads is required
What is the rational of using GnRH or its analogues?
- To either switch on or switch off the HPG axis
How do GnRH antagonists, native GnRH and GnRH agonists work?
1- Binds to GnRHR
2- Cell response = secretes LH/FSH
1- Bind and block GnRHR
- Competitive inhibitor of the GnRHR - compete with GnRH for the same receptor
1- Bind to GnRHR
2- Initial response = FSH/LH secretion
3- After a while, shut down of HPG axis
What are the characteristics of native GnRH?
- Synthetic GnRH - same primary sequence as endogenous GnRH
- Deca-peptide (Glycinamide group at position 10)
- Pulsatile mode of delivery = switching on
Why do we need GnRH analogues?
- GnRH t1/2 in circulation is 2-4 mins
- To increase potency & duration of GnRH → analogues created ⇒ agonists or antagonists
- Manipulate the HPG axis in clinical practice- useful in IVF, Hormone responsive cancers, endometriosis
What is the structure of GnRH?
What sequence is highly conserved in all mammals and most species?
- AA in positions 1-4 & 9-10 & Amide group
- Important residues for GnRHR binding & Activation
Describe the function of each section of the GnRH structure?
- Horseshoe conformation after undergoing transcribing translation and protein folding.
- Position 6: Agonist formation when Glycine undergoes D-AA substitution
- What are D-Amino Acids?
- Stereoisomers of L-AA
- E.g. D-Arg is the stereoisomer of L-Arg
How are GnRH agonists synthesised?
- Substitution of Gly by D-amino acids (Position 6)
- Replacement of Gly-NH2 by NH2-ethylamide binding to Pro (pos 9/10) for increased stability and resistance to proteolytic cleavage.
Give 2 examples of GnRH agonists
- Lupron: D Leu substituted + NEt
= 10 fold increase in GnRH activity (10x more potent than endogenous GnRH and 10x more GnRHR activity) - Buserelin: Most popular agonist used in IVF; D Ser sub + Net
= 100x increase in GnRH activity
What is the advantage of NEt substitution in positions 9/10?
- Allows GnRH agonist to avoid proteolytic cleavage
- exogenous injection = higher risk of digestion by proteases and thus risk of being broken-down.
History of GnRH antagonists creation and how are they made now?
- 1st gen replaced His & Trp at pos 2 & 3, but low suppressive activity
- 2nd gen potency increased by D-aa substitution in pos 6 but anaphylaxis by histamine release
- 3rd gen replaced D-Arg by D-ureidoalkayl aa
What were the issues with the 1st and 2nd gen of GnRH antagonists?
- 1st: low suppressive activity - function of antagonist is to suppress!!
- 2nd gen: Many patients had anaphylactic reactions thus withdrawn from market
Give examples of GnRH antagonists and where the changes in the structures are
What is the aim of antagonists?
- To maintain high binding affinity
- And block GnRHR activation
Mechanisms of action of GnRH and GnRH analogues
Describe the desensitisation of the GnRHR?
- The sustained agonist exposure to GnRHR
- Downstream pathways of the receptor are uncoupled
= Shut down of HPG axis - reversible
Clinical uses of native GnRH
- To diagnose & treat HH
What is 1° hypogonadism? What do these individuals present with?
- arises from gonadal failure
- primary gonadal failure -> Gonads produce little or no steroids = little or no steroidal feedback.
- Individuals with 1° hypogonadism present with low levels of gonadal steroids & NORMAL TO ↑ levels of FSH/LH.
What is 2° hypogonadism
- arises from abnormalities of hypo-pituitary axis
- hypothalamic or pituitary failure -> downregulation /shutdown of gonadotrophin release = ↓gonadal function -> gonads produce ↓ or no steroids = ↓ or no steroidal feedback.
- Individuals with 2° hypogonadism present with ↓ levels of gonadal steroids & ↓ levels of FSH/LH.
Give an example of using native GnRH for a diagnostic test
- To diagnose and treat Hypogonadotrophic Hypogonadism (HH)
- To distinguish between 1° & 2° hypogonadism
- Test: GnRH is administered intravenously or subcutaneously and plasma LH and FSH are measured at 0, 15, 30, 45 and 60 minutes
-> High FSH/LH = 1°
-> Low FSH?LH = 2°
What is Hypogonadism?
- Hypogonadism = impaired gonadal function with resultant decreased sex steroids
How is HH treated using native GnRH?
- Pump attached to patient -> GnRH provided in a pulsatile manner
What is the issue with diagnosing HH?
- Difficult to distinguish between delayed puberty & HH because pre-pubertal pituitary is unresponsive
Characteristics of delayed puberty
- Boys: testicular growth (volume >4 ml) has not started at 14yrs
- Girls: breast development is not present at 13yrs or menarche did not occur 15-18 years of age
Clinical uses of GnRH analogues
- IVF
- Hormone-dependent cancers: BC & PC
- Dysfunctional uterine bleeding
- Precocious puberty
- Hirsutism & virilisation
- Endometriosis
How is the HPG axis manipulated in IVF?
1 - Continuous administration of GnRH agonist/antagonist = HPG axis shuts down
2 - Exogenous administration of gonadotrophins
= stimulates follicular growth + recruits astral follicles
3 - Follicles tracked -> 3x 16-18mm in size = ready for oocyte retrieval
4- Administration of hCG
5- 36-48hrs later - oocytes retrieved
How is the HPG axis manipulated in IVF?
1 - Continuous administration of GnRH agonist/antagonist = HPG axis shuts down
2 - Exogenous administration of gonadotrophins
= stimulates follicular growth + recruits astral follicles
3 - Follicles tracked -> 3x 16-18mm in size = ready for oocyte retrieval
4- Administration of hCG
5- 36-48 hrs later - oocytes retrieved
What are we trying to achieve during IVF when shutting down the HPG axis?
- Take control of ovaries to stimulate them to recruit multiple oocytes
- thus we shut down HPG axis to allow this ovary control
How long does it take to see the effects of GnRH agonists vs antagonists during IVF?
- Agonists: 1-2 weeks
- Antagonists: 7 days
How are follicles tracked during IVF?
- The size of the follicles are tracked (ultrasound)
- Blood E2 levels tracked
- At least 3 Follicles that are 16-18mm in size => oocyte retrieval
What is the significance of administering hCG during IVF?
- Triggers final maturation of the oocytes
- Thus, triggers ovulation
Why is oocyte retrieval done within 36-48 hours?
- Later than this, ovulation takes place
- Thus, loss of oocytes
Why do we use hCG in IVF?
- LH-like properties
- Longer half-life than LH
- More stable than LH in its recombinant form
- Can bind & activate LHR => replaces LH in order to trigger ovulation
Why do we use hCG in IVF?
- LH-like properties
- Longer half-life than LH
- More stable than LH in its recombinant form
- Can bind & activate LHR => replaces LH in order to trigger ovulation
Steps in IVF?
1- Oocyte retrieval: Ultrasound guidance- ultrasound probe attached to aspirator needle which punctures follicle & drains follicular fluid & oocyte (collected in tube)
2- IVF: oocytes in culture media & insemination of oocytes approved to move onto next stage
3- Embryo transfer
How long do we culture the resulting embryo in IVF?
5 days
- day 0 to day 5 (after which is transferred to uterus)
What stage are the majority of oocytes in?
- MII
- Some MI -> leave for a few hours -> MII stage
What is used to stimulate follicle growth in IVF?
- GnRH agonist
- Gonadotrophins
Benefits of using GnRH agonists(& exogenous gonadotrophins) during IVF
- improved follicular recruitment = larger no. oocytes recovered (not in all patients)
- prevent premature LH surge (i.e. no shut down -> LH surge= ovulation of follicles) = lower cancellation rate
- Improvement in routine organisation
How are GnRH agonists used in breast cancer treatment?
Bc cells= E2 dependent -> shut down of HPG axis = shut down of E2 production -> growth of BC due to E2 ceased
- In premenopausal women -> GnRHa causes chemical castration (due to reduced E2 output)
- GnRHR present in BC tissue (50-60%) = direct anti-proliferative effect of GnRHa in BCa cell lines
What is chemical castration?
- using drugs to stop sex hormone production
How are GnRH agonists used in prostrate cancer treatment?
- 80% of PCa are androgen-dependent
- GnRH agonist → desensitisation →↓↓ T (chemical castration)
- Takes 7-10 days
- “Flare-effect” = ↑T (∵ ↑ FSH/LH initially, before it shuts down)
- Micro-surges of T, LH & FSH with continued use
- THUS co-administer GnRHa with anti-androgens (blocks AR & prevents T from activating its receptor = prevent flare effect)
Why do a large % of BC patients develop premature ovarian failure (POF)?
- ∵ follicular damage
- Chemotherapeutic agents directly attack DNA in dividing and dormant germ cells
How can we use GnRH agonists for fertility preservation in BC patients?
-Administer GnRHa to minimise gonadal damage
To preserve fertility either :
- Cryopreserve embryos or MII oocytes after IVF & before chemotherapy
- Cryopreserve ovarian tissue for transplantation later
Characteristics of the study that suggested how to preserve fertility in BC patients
- Xu et al (2011) Nature Medicine 17:1562-63
- Study in mice
- In humans, this method was mainly refuted
Limitations of GnRHa?
- Temporary solution - symptoms can return
- Side-effects: pseudo-menopause (hot flashes, insomnia, ↓ libido) in women (with associated symptoms): ↓ libido, erectile dysfunction, ↑LDL/↓HDL cholesterol, insomnia, headaches
- Extra pituitary sites of action (e.g. oocyte, embryo, uterus) in animals, thus extra affects as GnRHR in others so may cause other unwanted affects
- “Flare effect” - before providing relief
- Chronic treatment (>6 months): Osteoporosis(E2 maintains bone density), Heart disease (HDL/LDL levels)
GnRH antagonists & their affects in prostate cancer treatment
- No “flare” or micro-surges.
- ↓ testosterone to castrate levels by day 3.
2 examples of GnRH antagonists used in PC treatment
- Abarelix (1st antagonist) withdrawn ∵ systemic allergic reaction (anaphylactic reaction).
- Degarelix -> rapid & sustained ↓ in Testo & PSA (prostate-specific antigen), used only in special causes of advanced prostate cancer.
Are agonists mainly used or antagonists for P cancer treatment?
- GnRH Agonists
Time it takes for GnRH agonists to apply their effects in PC treatment vs antagonists?
- Agonists: 7-10 days
- Antagonists: 3 days
Advantages of GnRH antagonists
- 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.
Disadvantages of GnRH antagonists
- Limited licenses available for wider use (highly controlled treatment regime)
- More expensive than agonists.
- Need ↑ dose than agonist 100mg/month versus 3-5mg/month (need ↑ levels of GnRHa ∵ competitive inhibitor).
- Competitive inhibitor, ∴ ↓ effective over time (once effect seen i.e. once R blocked, nothing more can be done, that is as good as an effect as possible).
How do we use native GnRH to diagnose primary/secondary hypogonadism?
- 1° hypogonadism: GnRH test administered (functional pituitary & hypothalamus) -> primary gonadal failure & no steroid production/feedback, you should expect to see an ↑ FSH/LH response.
- 2° hypogonadism: GnRH test (difficult to distinguish between hypothalamic & pituitary disorders using GnRH test alone). Key observation = ↓ FSH/LH response. The exact nature of the secondary hypogonadism (hypothalamic or pituitary) is usually confirmed using other clinical diagnostic measures (e.g. assays of other pituitary stimulating hormones; neuroimaging etc.).
