GnRH Analogues

Question 1

What property is taken advantage of to manipulate the GnRH axis using GnRH or GnRH analogues?

Answer 1

• When thinking about the therapeutic uses and therapeutic applications of GnRH and GnRH analogues, the one property that we take advantage of is the pulsatile versus continuous administration or effects of GnRH. When selectively manipulating the GnRH axis using GnRH or GnRH analogues, this is the property taken advantage of.
• Data shows pulsatile secretion/administration of GnRH results in upregulation of LH and FSH. Once that administration is switched to continuous, it is shut down. When reverting back to pulsatile, it is accompanied by that characteristic rise in LH and FSH. This is established.

Question 2

How does administration affect GnRH action?

Answer 2

1) Continuous low-dose/single high-dose of GnRH = Shutting down of HPG axis
- Downregulation of gonadotrophin secretion
- This is a crucial property that is taken advantage of when gonadal inhibition required, i.e. ‘selective medical hypophysectomy’
- A selective medical hypophysectomy is the equivalent of removing/taking the hypothalamus out of the picture with the pituitary. It is a continuous low dose or a single high dose (not a pulse) to shutdown the HPG axis. The single high dose, in effect, remains in the circulation since there is so much of it. There is loads of GnRH still available and still bound to the receptor (same effect as a continuous low dose).
2) Pulsatile mode of delivery- Switching on
- Upregulation of gonadotrophin secretion
- Take advantage of this property when stimulation of gonads required

Question 3

What is the rationale for native GnRH versus GnRH analogues?

Answer 3

• “Analogues” refers to GnRH agonists and GnRH antagonists.
• In the case of native GnRH, it binds to the GnRH receptor. This stimulates the downstream cellular response, so signalling pathways that ultimately result in secretion of LH and FSH. In the case of GnRH agonists, they bind to the GnRH receptor and trigger the same response as native GnRH (initial transient expression of LH and FSH). However, this is only short-lived. After a while, that response is then terminated and there is no downstream response. The agonist binds to the GnRH receptor and initially has the same effects as GnRH. The difference is that GnRH has a short half life. It is degraded and leaves the receptor, hence the pulses. GnRH agonist stays there, on the other hand, so there is desensitisation (not in the classical sense). Hence, that GnRH response is terminated. There is no FSH and LH secretion anymore and the axis is shutdown (Agonist does not dissociate from receptor; is just there).
• In the case of the antagonist (competitive inhibitor), the antagonist just binds to the receptor and blocks it. There are no downstream effects. The agonist is an initial activator that inhibits by desensitisation, while the antagonist inhibits straight away.
• In the case of native GnRH and GnRH agonists, there is pulsatile GnRH function (switching it on). The inhibition in the case of antagonists is switching off/shutting down the HPX axis. Native GnRH is the exact same sequence and the exact same protein as what is produced in the hypothalamus but, in the case of clinical application, this is usually synthetically produced. It is the exact same thing, it is just recombinant, synthetic GnRH (same sequence, same protein).

Question 4

What is native GnRH?

Answer 4

• Synthetic GnRH- same primary sequence as endogenous GnRH
• Pulsatile mode of delivery →
  Switching on of HPG axis
• Looking at the structure of native GnRH, a decapeptide with an amide group attached to glycine at position 10, it is highly conserved across vertebrates except for a single amino acid substitution
• Synthetic GnRH with the exact same sequence as endogenous GnRH.

Question 5

Why do we need GnRH analogues if we are able to synthesise GnRH at the pharmaceutical level?

Answer 5

• GnRH has a very short half-life; degraded shortly in the system after released. When administering something exogenously on a pharmaceutical level, something with a longer half life that can last longer and modulate the desired effects for a longer period of time is required. Need to increase the potency and duration of GnRH. This is why GnRH analogues were created.
• 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- IVF, Hormone responsive cancers (particularly breast and prostate), endometriosis (treating endometriosis symptoms).
• Selective manipulation of the HPG axis is required in IVF, so GnRH agonists and sometimes antagonists are used to shut down the axis.

Question 6

What regions of the GnRH structure are highly conserved?

Answer 6

• GnRH = Glu, His, Trp, Ser, Tyr, Gly, Leu, Arg, Pro, Gly-amide
• Decapeptide with an amide group attached to glycine in position 10. Highly conserved. This configuration is crucial for GnRH binding and activation of the receptor. When looking at the creation of agonist and antagonists, there is a lot of manipulation that takes place in the sequence. The amino acids in positions 1-4 and 9-10 (with the amide group attached) are the highly conserved areas. The amino acids substitutions rarely occur in these bits; these bits are crucial for that binding and activity of GnRH.
• Highly conserved in all mammals and most species- = important residues for GnRHR binding and activation
• Positions 1 to 3 in particular (may include 4) are crucial for receptor binding and activation. Positions 8 to 10 are also very crucial for receptor binding. Position 6, which is always glycine, is crucial for stability and activity of GnRH. The arginine in position 8 is where it is most variable across species.
• Amino acid substitutions = one substitution that differentiates the form of GnRH that is being expressed across vertebrates; usually found at position 8 (this is where the difference between the different forms of GnRH being expressed is found).

Question 7

How is the structure of GnRH manipulated?

Answer 7

• GnRH = Glu, His, Trp, Ser, Tyr, Gly, Leu, Arg, Pro, Gly-amide
• When GnRH has been since synthesised and protein folding has taken place, it usually takes this horseshoe configuration (following the posttranslational modifications and protein folding).
• Positions 1 to 3 in particular (may include 4) are crucial for receptor binding and activation. Positions 8 to 10 are also very crucial for (only) receptor binding. Position 6, which is always glycine, is crucial for stability and activity of GnRH. The arginine in position 8 is where it is most variable across species. Amino acid substitutions = one substitution that differentiates the form of GnRH that is being expressed across vertebrates; usually found at position 8 (this is where the difference between the different forms of GnRH being expressed is found).
• When talking about the manipulations and changes that are being made to create analogues, D-amino acid substitutions are being made in these areas which are crucial for receptor binding and activation (positions 1 - 3) in antagonists. The amino acids present are replaced with D-amino acids. This is also done in position 6; the glycine is replaced with a D-amino acid to enhance the activity and stability.
• A D-amino acid is a stereoisomer of naturally occurring amino acids (L-amino acids); can be a D- isomer of another amino acid. This is done to create analogues, agonist and antagonists.

Question 8

How are GnRH agonists made?

Answer 8

• GnRH = Glu, His, Trp, Ser, Tyr, Gly, Leu, Arg, Pro, Gly-amide
• Straightforward to make agonist
  1) Substitution of Gly by D-amino acids
  2) 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
• All agonists & antagonists have substitution of Gly with D-aa at position 6 → stabilises conformation & enhances activity
• Making agonists was quite straight forward; a lot of the focus was on position 6. They substituted glycine with a D-amino acid. In some cases, they replaced the glycine amide group in position 10 with an ethylamide group. Those were the key changes that were made in the case of agonists.

Question 9

What GnRH agonists are available on the market as injectables?

Answer 9

• GnRH = Glu, His, Trp, Ser, Tyr, Gly, Leu, Arg, Pro, Gly-amide
• The different proprietary brands can be compared to the native GnRH structure.
• In the case of Lupron, a popular brand of GnRH agonist that is used clinically in IVF, glycine was replaced in position 6 with D-leucin and the glycinamide was replaced with an ethylamide group instead. Those two changes result in a tenfold increase in GnRH activity. Looking at Buserelin, the most popular GnRH agonist in IVF, glycine was replaced with D-serin and an ethylamide. This resulted in a 100 fold increase in GnRH activity. Another added benefit of the manipulation of the GnRH structure is that it helps avoid proteolytic cleavage. Once these agonists (peptides) are introduced into the system, naturally, there is risk of it being digested by proteases/broken down by enzymes. These changes make them resistant to proteolytic cleavage, so they can go on and exert the function they were designed for.
• Buserelin and Lupron can be self administered; in the case of IVF, the patient would inject themselves daily with the agonist. The aim is to shutdown the HPG axis and take control so FSH can be administered to stimulate follicle growth.
• Lupron (TAP), Zoladex (Zeneca), Supprelin (Roberts) and Synarel (Searle) result in a 10 fold increase in GnRH activity.
• Triptorelin (Ferring) and Buserelin (Hoescht) result in a 100 fold increase in GnRH activity.

Question 10

How are GnRH antagonists made?

Answer 10

• GnRH = Glu, His, Trp, Ser, Tyr, Gly, Leu, Arg, Pro, Gly-amide
• 30 years to make antagonist!
  1) 1st generation replaced His & Trp at pos 2 & 3 with D-amino acid substitutions, but low suppressive activity
  2) 2nd generation potency increased by D-aa substitution in pos 6 but anaphylaxis by histamine release (had to be withdrawn from market)
  3) 3rd generation replaced D-Arg by D-ureidoalkayl aa. This made the difference
• Antagonists were a lot more difficult to make; required a lot more manipulations of the GnRH structure.
• Took that long to make because of the anaphylaxis that was occurring.
• These replacements are usually in the first portions of our positions, 1 to 3, and also 5 to 10.

Question 11

What GnRH antagonists are available?

Answer 11

1) Cetorelix (Asta Medica)
2) Ganirelix (Organon)
3) Abarelix (Praecis)
4) Antide (Ares Serono)
5) Teverelix (Ardana)
6) FE 200486 (Ferring)
7) Nal-Glu (NIH)
- Maintains high binding affinity, blocks GnRHR activation
- There are a lot more manipulations
- In all of these proprietary brands, positions 1 to 3 have all been swapped. There is a change in position 6, in some of them there’s a change is well in position 8 and all of them have changes in position 10 as well. There is a lot more manipulation that takes place in the case of antagonists. This maintains high binding activity, because the antagonists are essentially competing with GnRH for the receptor. They require high affinity to reach the receptor ahead/instead of GnRH to enable it to block the receptor. This is the effect of all of these changes.

Question 12

What is the mechanism of action of GnRH and GnRH analogues?

Answer 12

• In the case of native GnRH, it binds to the receptor, there is activation of downstream signalling (transcription of FSH and LH), this stimulates the secretion of FSH and LH and then the GnRH ligand is dissociated from the receptor. The receptor becomes responsive (awaits) the next pulse. This is down to the short half-life of native GnRH.
• In the case of the agonist, it also binds to the receptor, there is the same activation of signalling, stimulation of FSH and LH synthesis and secretion, but then there is a desensitisation of the GnRH receptor.
• The GnRH receptor is resistant to the classical desensitisation. In this case, when the agonist remains on the GnRH receptor, the Gs and Gq pathways get uncoupled from the receptor. This is how it is believed this form of desensitisation occurs (rather than the classical way of the C terminal tail being phosphorylated and the receptor being internalised and becoming unavailable to GnRH). The receptor remains intact with the agonist bound to it. The only thing, in this case, is that the GnRH receptor uncouples from the Gs and Gq pathways, so GnRH receptor becomes non-responsive. Since desensitisation does not occur in the classical sense because there is no C terminal tail in the receptor, while the agonist remains bound to the receptor, the Gs and Gq disengage. Normally, the natural response would be for Gs and Gq to be activated once GnRH binds to the receptors. However, once the agonist remains after that initial activation, then the Gs and Gq just disengage and uncouple from the receptor. This is what is believed to be the mode of desensitisation based on the evidence that we have now.
• In the case of antagonists, they bind to the receptor, block it and there are no downstream effects. These are the differences between the three.

Question 13

What are the advantages of GnRH antagonists?

Answer 13

• 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” (in the case of both IVF and cancers).
• Dose-dependent; Partial pituitary-gonadal inhibition, Can adjust level of hypogonadism as desired.
• Can also manipulate the dose; can adjust the level of HPG axis downregulation by adjusting the dose of antagonist.

Question 14

What are the disadvantages of GnRH antagonists?

Answer 14

• Limited licenses available for wider use (only used in very specific cases).
• More expensive than agonists. More expensive to make and more expensive for patients.
• Need higher dose than agonist 100mg/month versus 3-5mg (it must compete with GnRH to block the receptor, so a stronger and more potent dose is required to achieve this).
• Competitive inhibitor, therefore less effective over time. Once you have succeeded in blocking the receptor and you have seen the effects of that, there is only so much that can be done after. Nothing more can be done after the receptor has been blocked.

Question 15

A prostate cancer patient is administered a treatment regimen containing a combination of Lupron (GnRH analogue) and Flutamide (androgen receptor antagonist)

1) What class of GnRH analogues does Lupron belong to and what is its mechanism of action?
2) Why was Flutamide prescribed to this patient?

Answer 15

1) Lupron is an agonist; shuts down the HPG axis. It binds to the GnRH receptor, causes normal activation of signalling that stimulates LH and FSH secretion. It stays bound, so there is eventual desensitisation of the GnRH receptor (Gs and Gq pathways become uncoupled) and it stops responding to GnRH (receptor is ultimately inactive).
2) To prevent the flare effect; prevents testosterone from binding to its receptor.

Question 16

What are the clinical uses of native GnRH and GnRH analogues?

Answer 16

• Native GnRH
  1) Pubertal blockers for potential gender reassignment or sex change
  2) Diagnostic tests
  3) Hypogonadotrophic hypogonadism (HH)
  4) Delayed puberty
• GnRH Analogues
  5) IVF
  6) Dysfunctional uterine bleeding
  7) Precocious puberty
  8) Hormone-dependent cancers = Breast cancer, Prostate cancer
  9) Hirsutism and virilisation
  10) Endometriosis
• Looking closer at analogues, the most popular uses of GnRH analogues have been highlighted (most popular = IVF)
• IVF is a billion-dollar industry worldwide. Lots of analogues are key to the treatment regimen in IVF.
• Hormone-dependent cancers are also a very common clinical application

Question 17

How are GnRH analogues used clinically as pubertal blockers for potential gender reassignment or sex change?

Answer 17

• In the case of pubertal blockers for potential gender reassignment or sex change, analogues are used to shutdown the HPG axis so the relevant hormones required can be administered to bring about the secondary sex characteristics that are designed.

Question 18

How is native GnRH used clinically in diagnostic tests?

Answer 18

• Hypogonadism defined as impaired gonadal function with resultant decreased sex steroids
• To distinguish between 1° & 2° hypogonadism
• 1∘ hypogonadism arises from gonadal failure (testicular/ovarian failure
• 2∘hypogonadism arises from abnormalities of hypo-pituitary axis
• Test: GnRH is administered intravenously or subcutaneously and plasma LH and FSH are measured at 0, 15, 30, 45 and 60 minutes.
  Condition can arise from gonadal failure (primary hypogonadism) or abnormalities of hypoth-pit axis (secondary hypogonadism). Distinction is usually made by measurement of LH & FSH.
• Gonadotropin deficiency, may be seen in patients with: Large pituitary tumors, Endocrine deficiency, Hemochromatosis, Kallmann syndrome, Hyperprolactinemia , Amenorrhea, Anorexia nervosa, Starvation
• Diagnostic: FSH & LH is measured before and x4 in hour after administration of GnRH injection. Primary hypogonadism starts in ovary/testes- will have low levels of gonadal steroids along with high levels of LH & FSH and secondary hypogonadism indicates problem in hyp/pituitary axis.
  A normalish FHS/LH response suggests that gonadal failure is due to a problem within the ovaries or testes. (i.e. GnRH is able to elicit LH response, hence reason gonads not functioning is not because not receiving gonadotrophins but because not producing/responding to gonadal steroids). If the response is excessive could indicate hypothalamic dysfunction
  A reduced FSH/LH response suggests a problem with the hypothalamus or pituitary gland. However very difficult to interpret and if trying to diagnose at puberty then levels of gonadotrophins will depend on stage of puberty.
• Native GnRH is a useful diagnostic tool that can be used to distinguish between primary and secondary hypogonadism.
• Take blood tests intermittently an assay GnRH levels
• GnRH is produced in the hypothalamus, the anterior pituitary response with upregulation of LH and FSH and the steroid hormones from the gonads feed back. When there is gonadal failure, primary hypogonadism, there is no gonadal steroid feedback, so there is an increase in FSH and LH production. In secondary hypogonadism (anterior pituitary/hypothalamic abnormalities), the gonads are functioning but there is no proper function (failure) of the hypothalamus or anterior pituitary so there is no upregulation of FSH and LH (downregulation). This is the difference between primary and secondary hypogonadism.
• GnRH is administered intermittently at specified periods and serum levels of LH and FSH are tested. In the case of primary hypogonadism, high levels of FSH and LH are seen. In the case of secondary hypogonadism, low levels of LH and FSH are seen. This is how GnRH is used as a diagnostic tool. However, it is important to know that it can be difficult to distinguish between pubertal disorders like these and constitutive delay in puberty. In a situation where a prepubescent individual that just has a constitutive delay (just a natural delay that would eventually come to an end so the individual would undergo puberty) due to metabolic status or gene expression, for example, GnRH is not that useful; no response would be seen. The pituitary gland of a prepubescent individual is non-responsive to GnRH

Question 19

How is native GnRH used clinically to diagnose and treat hypogonadotrophic hypogonadism?

Answer 19

• Used to diagnose hypogonadism. It can also be used to treat it as well, especially in the case of secondary. They attach a pump to the individual that would deliver GnRH in pulses with hope of activating the axis. In the case of delayed puberty, this is usually where the challenge is, especially if the delay is constitutive instead of being caused by other factors. Delayed puberty is defined by testicular growth and breast development in boys and girls, respectively.
• Delayed puberty =
  1) Boys, when testicular growth (volume >4 ml) has not started at 14yrs,
  2) Girls, when breast development is not present at 13yrs or menarche did not occur 15-18 years of age
• Tricky to distinguish between delayed puberty and HH because in pre-pubertal state, the pituitary will hardly respond. Pre-pubertal pituitary is unresponsive. Not much benefit from using native GnRH as a treatment.

Question 20

How is the HPG axis manipulated in IVF?

Answer 20

• In the normal HPG axis, there is pulsatile GnRH released by the hypothalamus onto the anterior pituitary. In turn, there is pulsatile expression of FSH and LH. As a result, mid-cycle, there is selection of the dominant follicle which is followed by ovulation. That single egg is ovulated and then the gonadal steroids feed back negatively.
• However, in the case of IVF, when the agonists are applied (continuous), there is a shutdown of the HPG axis. The archetypal menstrual cycle with dominant follicle selection is shutdown; uncouples the HPG axis by doing this. In some cases, antagonists are used as well, but it is usually agonists used in IVF.
• Once the HPG axis is uncoupled, a state of pseudo menopause ahs essentially been created by shutting down ovarian function. This allows the IVF clinic to introduce exogenous FSH to allow for recruitment of multiple follicles as opposed to the one follicle in a normal cycle. The reason for shutting down the HPG axis before doing this is because administering exogenous gonadotrophins without doing this stands the risk of generating multiple follicles and all of them being ovulated then lost. Also, there is going to be feedback that will counteract what is trying to be achieved with the ovaries.
• Once clinicians have tracked these follicles and have a good number of the required size, they trigger ovulation and the final maturation process by administrating hCG (usually the LH surge that triggers complete maturation and ovulation in the natural cycle). HCG has a longer half life and LH-like properties. HCG can bind and activate the LH receptor. It is more stable in the synthetic form than LH, so it is used in triggering ovulation and complete maturation over LH. Ocytes retrieval. Once the pregnancy has been established and the embryo is producing hCG, the hCG binds to the LH receptors on the corpus luteum to keep progesterone production going.

Question 21

What are the stages of IVF?

Answer 21

• First, hormonal stimulation is carried out; the HPG axis has been shut down with analogues and daily doses of FSH (and a bit of LH in some cases) have been administered. Once clinicians are happy with the number of follicles that they can see upon ultrasound tracking (at least three follicles between 16 and 18mm), then they proceed and schedule the patient for egg collection.
• While FSH administration is going on, they track the growth of the follicles with ultrasounds of the ovary and also tracking blood oestrogen levels. Blood samples are taken regularly to track oestrogen levels. The criteria varies from clinic to clinic, but generally requires about three of that size. HCG is then administered at that point to trigger completion of maturation. 36 hours later, oocyte retrieval is performed (time sensitive! If oocyte retrieval is not performed within that time period, it risks ovulation happening by itself and eggs are lost! There is also risk of multiple pregnancy if the women were to have unprotected intercourse!). Oocyte retrieval is carried out under the guidance of ultrasound probe connected to needle inserted in vagina. This needle, as guided by ultrasound, is used to puncture the follicles. The fluid in the follicle is aspirated and the oocytes are contained within this fluid (also aspirated along with the fluid).
• The ultrasound image shows large, dark holes. Those are the follicles that the clinician will see as they are performing the oocyte retrieval. Their job is to drain each one of the follicles that are visible. As that is being done as well, the follicular fluid containing the oocytes is passed to the clinical embryologist who examines the fluid under the microscope and look for the eggs in them. These oocytes are taken out of the follicular fluid and transferred into a culture in dishes. While this is happening, the embryologist is giving feedback to the clinician regarding how many oocytes were collected. If they had tracked an ultrasound that they were expecting six follicles to be of antral follicle size and they only see one egg after draining all of them, it allows the clinician to further investigate what may have been the case. It is not always a guarantee that if a certain number of follicles meet the criteria, the number of eggs will match, but it allows the clinician to pay closer attention to what may have occurred. Once the oocytes have been collected, they are left in culture for a few hours. Usually, it would be expected that those antral follicles are at the metaphase II stage (which is the stage where they are competent for fertilisation). Some of them might still be in metaphase I and leaving them for a few hours will allow them to cross over and complete the meiotic division. Once they have sat in culture, depending on the recommended treatment option, they will either have conventional IVF (sperm is mixed with oocytes in culture dish and left in incubator to fertilise by themselves) or, in the case of male factor infertility where the male partner sperm quality is below normal, there is a treatment option called ICSI (intracytoplasmic sperm injection; each individual sperm is injected into an individual egg). Once the fertilisation process has been carried out, by whichever method, select an incubator and assess fertilisation in the next morning. From there, they continue to monitor embryonic development in culture. Embryos are usually cultured up till day 5, which is the blastocyst stage. Day of oocyte retrieval = day 0, day of embryo transfer = usually day 5. The culture to blastocyst stage is usually dependent on the number and the quality. In the case where the patient just has one or two and they are not meeting those expected developmental targets (they’re not developing as they should), the recommended practice would usually be to return them to the mother as early as possible to give them a better chance. In those situations, they may have a day 3 transfer where the embryo is still at the 8-cell stage, or a day 2 transfer where the embryo is still at the 4-cell stage. Generally, we like to culture up to the blastocyst stage, which is day five.
• At the moment, in the UK, there is a push for a single embryo transfer (only one embryo is put back. Legally, however, you are allowed to have up to two. In cases where you are over the age of 41, you can have up to three (case by case basis). In the case of embryo transfer, embryos are loaded in a catheter with a drop of culture media. By ultrasound guidance, the embryos/blastocysts are inserted into the uterus and expelled from the catheter.

Question 22

What are the benefits of using GnRH agonists in IVF?

Answer 22

• GnRH agonist + gonadotrophins used extensively for follicle growth stimulation in IVF
• Major benefit:
  1) improved follicular recruitment → larger no. oocytes recovered (not in all patients)
• prevent premature LH surge → lower cancellation rate
• Improvement in routine organisation (can better schedule procedures, such as oocyte retrieval and embryo transfer, in the way that best suits the patient and clinic)
• Administration of the agonists and the gonadotrophins (FSH and, in some cases, LH) are used extensively for follicle growth stimulation in IVF. The benefit of this is improved follicular recruitment. IVF treatment is very expensive; The average IVF cycle costs around £6000 at the very minimum. There is NHS funding if certain strict criteria are met but even in the cases that qualify, most CCGs (NHS care commissioning groups) only fund one IVF cycles and subsequent cycles come out of pocket. Therefore, when carrying out an IVF procedure, it is important to give the patient the best chance of success and one of the ways to do this is to have a large starting number of oocytes. This is why stimulation with gonadotrophins is conducted after shutting down the HPG axis with the agonists. Having that large starting number of oocytes, as opposed to the one oocyte every month in a natural cycle, gives patients a much better chance of it working. Another benefit of shutting down the HPG axis is that it prevents the premature LH surge. When the HPG axis is functioning and FSH is being administered, there is going to be increased follicular activity in turn. These follicles will produce oestrogen which feeds back to the HPG axis. When oestrogen levels reach the threshold, there will be the switch to positive feedback causing the surge. During IVF, the oestrogen levels exceed the threshold but there is no premature surge because the HPG axis has been shut down.

Question 23

How are GnRH agonists used in breast cancer?

Answer 23

• Premenopausal women → chemical castration (reduce oestrogen output)
• GnRHR present in breast cancer tissue (50-60%); Direct anti-proliferative effect of GnRHa in BCa cell lines
• There is evidence for a direct inhibitory effect of GnRHa in cancer cells, e.g. post-menopausal women with breast cancer shown that GnRH analogues have direct anti-proliferative effects that are independent of actions caused by a reduction in sex-hormones. Evidence: presence of GnRHr in breast cancer tissue and also the demonstration of anti-proliferative action of GnRH analogues in breast cancer cell-lines. This has been shown to be true in a no. of cell lines of reproductive tract tumours including prostate, uterine and ovarian cancers.
  Even though practically no GnRH in general circulation (all confined to hypothalamic hypophyseal circulation), still have GnRHr on breast, ovary, prostate cancer tissue..significance?
• GnRH can be used in hormone-dependent cancers. In premenopausal women who are undergoing treatment for cancer, the use of GnRH agonists is the equivalent of chemical castration. The HPG axis is essentially shut down, thus reducing oestrogen output (a lot of breast cancers are oestrogen-dependent). This is the key benefit of using GnRH agonist.
• When carrying out research on cancer cell lines, about 50 to 60% of them express the GnRH receptor. There is a direct antiproliferative effect of GnRH on a lot of cancer cell lines based on in vitro studies that have been carried out. Some of this has also been shown clinically in vivo as well.

Question 24

How are GnRH agonists used in prostate cancer?

Answer 24

• Prostate Cancer (PCa) is 2nd most frequent tumour in men in West
• 80% of PCa are androgen dependent
• GnRH agonist → desensitisation →↓↓ T (chemical castration)
• “Flare-effect” results ↑T
• Micro-surges of T, LH & FSH with continued use
• Co-administer with anti-androgens
• Applying the agonist shuts down the HPG axis, the production of FSH and LH and, ultimately, the production of testosterone (a lot of these cancers are testosterone-dependent).
• The equivalent of chemical castration.
• The flare effect is the initial activation of FSH and LH by the agonist. It results in upregulation of testosterone. In the case of prostate cancer where it is testosterone-dependant, that is like adding fuel to the fire. Want to treat the cancer and suppress the growth so shutting down the HPG axis by applying agonist but the initial flare effect is not a desired outcome. This is why it is co-administered with antiandrogens. When agonists are administered in the case of prostate cancer, antiandrogens are also administered to prevent the effects of testosterone in this case. Antiandrogens usually come in the form of androgen receptor blockers (essentially androgen receptor antagonists). They bind to the androgen receptor, in this case the testosterone receptor, and block it. The upregulation of testosterone (in the flare-effect) has no effect.
• When taking anabolic steroids, a lot of them are broken down into testosterone in the bloodstream. This abundance in serum levels of testosterone causes constant negative feedback on the brain. The testosterone brings about the desired characteristics, e.g. muscle build, but the detrimental effect is that endogenous testosterone production by the testes is shut down. Overtime, testicular atrophy (shrinking) begins to occur; becomes irreversible after a while and can cause infertility.

Question 25

How are GnRH agonists involved in fertility preservation (in cancer patients)?

Answer 25

• 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. As a result of cancer treatments, a lot of women develop premature ovarian failure due to damage that comes from chemo and radiotherapy.
• To preserve fertility either
  1) Cryopreserve embryos or MII oocytes after IVF and before chemotherapy. Now an option on the NHS.
  2) Cryopreserve ovarian tissue for transplantation later
• Administer adjuvant therapy to minimise gonadal damage?
• Prepubescent females (girls with childhood cancer) cannot benefit from these fertility preservation options. It is now believed that GnRH agonists can be used as a fertility preservative; adjuvant therapy to minimise damage - administering GnRH agonist before undergoing chemotherapy/cancer treatment would minimise the damage being done to the ovaries.
• This was based on this study conducted in mice. There was a control mouse that didn’t have any agonist administered and was put through a round of chemotherapy. As a result, follicular damage meant there were no viable follicles, resulting in premature menopause and infertility. In the mice that had GnRH agonist administered prior to chemo, they were able to retain a lot of viable follicles and, after the chemo, they were able to have their normal ovarian function restored and retained their fertility (viable follicles). It is based on studies like this that some clinical spaces believe that this is a useful treatment option. It has been highly refuted clinically in mainstream research, but there are still some clinics that like to offer this as a treatment. They believe that shutting down the HPG axis would make the ovaries and the follicles resistant to the harmful effects of the chemo.
  The best option till date is doing a round of ovarian stimulation, collecting the oocytes and freezing them.

Question 26

What are the limitations of GnRH agonists?

Answer 26

1) Temporary solution - symptoms can return in the case of cancers
2) Side-effects -pseudo-menopause in women (with associated symptoms) = reduced libido, erectile dysfunction (males with prostate cancer), increased LDL / decreased HDL cholesterol, insomnia, headaches
3) 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. - The GnRH receptor is known to be found in places other than the pituitary. The agonist is likely to be exerting actions on those extra pituitary sites as well. This is something that hasn’t been fully characterised, so we don’t know precisely what is going on in those regions.
4) “Flare effect” – initial upregulation of steroids (can encourage cancer growth)
5) Chronic treatment (>6 months). Long-term treatment with agonists. Osteoporosis from loss of oestrogen (needed to maintain bone density), Heart disease (changes in the serum lipids and cholesterol; has been linked with susceptibility).

Question 27

How have GnRH antagonists been used in cancer?

Answer 27

• In the case of antagonists for cancer, there are not many pharmaceutical licences out there; it is still an area that is being researched. Agonists are the more popular analogue of choice.
• So far, there is one proprietary brand that is quite common in the case of prostate cancer. Antagonists are not commonly used, but there is a brand called Degarelix which is routinely used in the advanced cases of prostate cancer (usually, the first port of call would be an agonist). The advantage of antagonists is that there are no flare effects or micro-surges in testosterone. It is very rapidly acting; reduces testosterone levels by day 3, which is a lot shorter than what happens with an agonist (at least a week). The reason why there are not that many licences available is similar to the research and development journey of IVF, where earlier versions had to be withdrawn due to adverse allergic reactions.
• Prostate Cancer
  1) No “flare” or micro-surges.
  2) Reduces testosterone to castrate levels by day 3.
  3) 1st antagonist Abarelix withdrawn due to systemic allergic reaction.
  4) Degarelix → rapid & sustained reduction in Testo & PSA (prostate specific antigen) routinely used now in advanced prostate cancer.