Drugs used for ovarian stimulation:

Clomiphene citrate
Introduction
Drug description
Mechanism of action
Duration of treatment
Side-effects and safety

Metformin
The drug
Pharmacokinetics
Side-effects

Human chorionic gonadotropin (hCG)
Pharmacokinetics
Dose of treatment
Recombinant drug

Gonadotropins
Historical overview
Human menopausal gonadotropin (hMG)
Purified follicle stimulating hormone
Purification of hMG
Recombinant human gonadotrophins (FSH, LH, hCG)
Quantifying and standardizing gonadotropin content
Molecular modeling and the development of ‘designer’ gonadotropins

gonadotropin-releasing hormone analogs
Introduction
GnRH agonists: Biosynthesis and mechanism of action
Side effects
Teratogenic effects
Gonadotropin-releasing hormone antagonist: Mechanism of action
Synthesis of GnRH antagonists
Safety and tolerability studies

Clomiphene citrate (CC)
Clomiphene citrate is one of the most popular drugs for ovulation induction because it is easy to administer, highly effective, and considerably safe, there is no need for close monitoring, and the cost is minimal. In the early days of IVF treatment with CC was very common; however, other mode of treatment replaced CC with more potent treatment.

drugs1

Drug description

Clomiphene citrate was synthesized in 1956, and an indisputable therapeutic breakthrough occurred in 1961 when Greenblatt and his group discovered that CC, a non-steroidal analog of estradiol, exerts a stimulatory effect on ovarian function in women with anovulatory infertility. The drug was approved for infertility treatment by the United States Food and Drug Administration (FDA), in 1967.

Mechanism of action
Administration of CC is followed in short sequence by enhanced release of pituitary gonadotropins, resulting in follicular recruitment, selection, assertion of dominance, and rupture.
The principal mechanism of CC action is a reduction in the negative feedback of endogenous estrogens due to prolonged depletion of hypothalamic and pituitary estrogen receptors (ER). This action consequently leads to an increase in the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus into the hypothalamic–pituitary portal circulation, engendering an increase in the release of pituitary gonadotropins. Administration of a moderate gonadotropin stimulus to the ovary overcomes the ovulation disturbances and increases the cohort of follicles reaching ovulation.

drugs2

Duration of treatment
Clomiphene citrate increases secretion of FSH and LH and is administered for a period of 5 days. In women with normal cycles, administration of CC for more than 5 days resulted in an initial increase of serum FSH concentration that lasted for 5–6 days, followed by a decline in serum FSH levels, despite continuation of the drug, whereas LH levels remained high throughout the entire treatment period.

Side-effects and safety
The most common side-effects are hot flushes (10%), abdominal distention, bloating or discomfort (5%), breast discomfort (2%), nausea and vomiting (2%), visual symptoms, and headache (1.5%). A rise in basal body temperature may be noted during the 5-day period of CC administration. Visual symptoms include spots (floaters), flashes, or abnormal perception. These symptoms are rare, universally disappear upon cessation of CC therapy, and have no permanent effect.
Several reports have associated long-term (12 months) CC therapy with a slight increase in future risk of ovarian cancer (relative risk (RR) 1.5–2.5). Owing to these initial reports, the Committee on Safety of Medicines (CSM) in the UK advised doctors to adhere to the manufacturers’ recommendations of limiting treatment to a maximum of 6 months. However, this increased risk has not been confirmed by subsequent reports.

Several case reports have linked CC with congenital malformations, especially neural tube defects (NTDs). Data available on 3751 births after CC treatment included 122 children born with congenital malformations (major and minor), representing an incidence of 32.5/1000 births. This figure is within the range found among the normal population.

Metformin

The biguanide metformin (dimethylbiguanide) is an oral antihyperglycaemic agent widely used in the management of non-insulin-dependent diabetes mellitus. It is an insulin sensitizer that reduces insulin resistance and insulin secretion. Over the last few years there has been increased interest  on the use of metformin (at doses of 1,500-2,500 mg/day ) to increase ovulatory frequency particularly in women described as having Polycystic Ovary Syndrome. Both the ASRM Practice Committee (USA)  and NICE (UK) have made recommendations for its use in treating anovulatory PCOS.
Metformin is available in 500 mg, 850 mg, and 1000 mg tablets. An extended release form is available in 500 mg tablets.

Pharmacokinetics
Metformin is administered orally and has an absolute bioavailability of 50 to 60%, and gastrointestinal absorption is apparently complete within 6 hours of ingestion. Metformin is rapidly distributed following absorption and does not bind to plasma proteins. No metabolites or conjugates of metformin have been identified. Metformin undergoes renal excretion and has a mean plasma elimination half-life after oral administration of between 4.0 and 8.7 hours. Food decreases the extent of and slightly delays the absorption of metformin.

drugs3

Side-effects

In one US double-blind clinical study of metformin in patients with Type 2 diabetes, the most reported adverse reactions (reported in > 5% patients) following metformin use were Diarrhea (53%),  Nausea/Vomiting (25.5%) , Flatulence (12.1%) , Asthenia (9.2%), Indigestion (7.1%%) , Abdominal Discomfort (6.4%), Headache (5.7%)

Human chorionic gonadotropin (hCG)

Human chorionic gonadotropin: the luteinizing hormone surge surrogate
Owing to inconsistency of the spontaneous LH surge, in controlled ovarian stimulation, and its inefficacy in patients being treated with GnRH agonists, hCG has been uniformly adopted by all successful ovarian-stimulation programs to effect the final triggering of ovulation. When preovulatory follicles are present, administration of hCG is followed by granulosa cell luteinization, a switch from estradiol to P synthesis, resumption of meiosis and oocyte maturation, and subsequent follicular rupture 36–40 hours later. These processes will occur only if the follicle is of appropriate size and granulosa and theca cell receptivity is adequate, depending on LH receptor status.

Human chorionic gonadotropin has been used as a surrogate LH surge because of the degree of homology between the two hormones. Both LH and hCG are glycoproteins and both have almost identical beta subunits.   Most important, they have the same natural function, i.e. to induce luteinization and support lutein cells.
Almost universal use of GnRH agonists and pituitary desensitization protocols has made the fear of untimely LH surges relatively obsolete; hence hCG, the timing of the LH-like stimulus, has been given greater flexibility.

Pharmacokinetics
The plasma metabolic clearance rate of hCG is slower than that of LH, i.e. a rapid disappearance phase in the first 5–9 hours after intramuscular (i.m.) injection, and a slower clearance rate in the 1–1.3 days after administration. The calculated initial and terminal half-life of recombinant hCG is 5.5+1.3 and 31+3.0 hours, respectively, as opposed to 1.2+0.2 and 10.5+7.9 hours, respectively, for recombinant human LH, as determined after intravenous (i.v.) administration of the drugs. By day 10 after administration, <10% of the originally administered hCG was measurable.

The long serum half-life of hCG is likely to be an undesirable characteristic in clinical practice. Residual hCG may be mistaken for early detection of de novo synthesis of hCG by a newly implanted pregnancy. Additional consequences of hCG administration are the sustained luteotropic effect, development of multiple corpora lutea, and supraphysiologic levels of estradiol and P synthesis. Sustained high-level stimulation of the corpora lutea may lead to ovarian hyperstimulation syndrome, a major complication of gonadotropin therapy.

It was shown that there was no difference in cycle outcome with random timing of hCG administration over a 3-day period. Unfortunately, invalidation of the pituitary mechanism that releases us from an inappropriate LH surge has also made us completely dependent on hCG, with all its inherent problems, for the final stage of ovulation triggering.

Dose of treatment
Another issue requiring clarification is the minimal effective dose of hCG in order to trigger oocyte maturation and ovulation. In a study examining the minimal effective dose of hCG in IVF, dosages of 2000, 5000, and 10000IU of urinary hCG were administered to 88, 110, and 104 women, respectively. No differences in oocyte recovery were noted when comparing the groups that received 5000 and 10000IU. However, a significantly lower number of oocytes were aspirated in the 2000-IU group, compared with the 5000- and 10000-IU groups.

Recombinant drug
With the recent development of recombinant technology, recombinant hCG became available for clinical use, and appears to be as efficacious as urinary hCG with the benefit of improved local tolerance. Using recombinant hCG (r-hCG) in the IVF program administration of 250 micg r-hCG compared with 5000IU urinary hCG revealed that r-hCG is at least as effective as 5000IU of urinary hCG. Using a higher dose of r-hCG, such as 500 micg, resulted in retrieval of more oocytes, but a three-fold increase of ovarian hyperstimulation syndrome (OHSS) occurred. It was also noted that local reaction to the injection was significantly better than to the urinary product of equal dose.

Urinary Gonadotropins
Historical Overview

In 1927, Aschheim and Zondek discovered a substance in the urine of pregnant women with the same action as the gonadotropic factor in the anterior pituitary. They coined this substance gondadotropin or “prolan.” They subsequently used their findings to develop the pregnancy test that carries their names. In 1930, Zondek reported that gonadotropins were also present in the urine of postmenopausal women, and in the same year, Cole and Hart found gonadotropins in the serum of pregnant mares. However, it was only in 1937 that Cartland and Nelson were able to produce a purified extract of this hormone. It was not until 1948, as a result of the work of Stewart, Sano, and Montgomery, that gonadotropins in the urine of pregnant women were shown to originate from the chorionic villi of the placenta, rather than the pituitary. It was subsequently designated “chorionic gonadotropin.” Piero Donini, a chemist at the Pharmaceutical Institute, Serono, in Rome tried to purify hMG from postmenopausal urine. The first urine extract of gonadotropin contained LH and FSH and was named Pergonal®, inspired by the Italian words “per gonadi” (for the gonads). The approval to market Pergonal was first granted by the Italian authorities in 1950.
Only in 1961, with Pergonal treatment, was the first pregnancy achieved in a patient with secondary amenorrhea, which resulted with the birth (in 1962 in Israel) of the first normal baby girl.

Human menopausal gonadotropin (hMG)
Human menopausal gonadotropin contains an equivalent amount of 75IU FSH and 75IU LH in vivo bioactivity. It was also demonstrated that hMG preparations also contain up to five different FSH isohormones and up to nine LH species. These differences may cause discrepancies in patients’ responses occasionally observed when using various lots of the same preparation.
Follicle stimulating hormone, which is the major active agent, accounts for <5% of the local protein content in extracted urinary gonadotropin products. The specific activity of these products does not usually exceed 150IU/mg protein. The different proteins found in various hMG preparations include tumor necrosis factor binding protein I, transferrin, urokinase, Tamm–Horsfall glycoprotein, epidermal growth factor, and immunoglobulin-related proteins. Although hMG preparations are effective and relatively safe, local side-effects, such as pain and allergic reactions, that were possibly attributed to immune reactions related to nongonadotropin proteins, have been documented in certain cases. Information is scarce regarding the metabolism of gonadotropin hormones.
Measuring levels of gonadotropins by in vivo bioassays serves to compare biologic effects of gonadotropin preparations in a quantitative manner in animals. In the extensively used Steelman–Pohley assay,77 21-day-old female Sprague–Dawley rats are injected subcutaneously (s.c.) for 3 days and their ovaries weighed on the fourth day. Disadvantages of this assay are that its sensitivity is too low to detect small amounts of FSH in the serum, reproducibility is poor (+20% variation), and the procedure is cumbersome. The reliance on this assay, in effect, signifies that an ampoule of hMG, which appears to have 75IU of FSH, may actually contain between 60 and 90IU.Circulating levels of the gonadotropins measured at any given moment represent the balance between pituitary release and metabolic clearance. After i.v. injection, the initial half-life of urinary FSH was demonstrated to be approximately 2 hours, and the true terminal (elimination) half-life appeared to be 17+5 hours. After i.m. injection of urinary FSH preparations, the half-life was estimated to be approximately 35 hours.

Purified follicle stimulating hormone
Urinary FSH  and highly purified FSH became available with the development of new technologies using specific monoclonal antibodies to bind the FSH and LH molecules in the hMG material in such a way that unknown urinary proteins could be removed.
Further purification of hMG substantially decreased LH-like activity, leading to a commercial pFSH preparation. Metrodin was introduced in the mid-1980s and is a product from the same source as hMG, but the LH component has been removed by immunoaffinity chromatography.
Apart from obtaining a more purified product, the rationale of developing a pFSH preparation was that ovulation induction using gonadotropins in patients with elevated endogenous LH serum levels could, on theoretical grounds, preferably be performed without exogenously administered LH. It was also suggested that FSH alone could increase folliculogenesis. Furthermore, it was speculated that LH in gonadotropin preparations could be responsible for the high incidence of complications in patients with elevated serum LH levels. However, other studies have indicated that the effectiveness of gonadotropin preparations and the occurrence of OHSS were not dependent on the LH:FSH ratio, albeit the administration of pFSH to patients with PCOS did result in decreased LH levels, compared with hMG.
The desirable goal of having an FSH preparation of high purity led to the development of an immuno-purified product (Metrodin-HP) of >95% purity.
Metrodin has a specific activity of 100–200IU of FSH/mg of protein, whereas Metrodin-HP highly purified has an activity of approximately 9000IU/mg of protein.

Purification of hMG
A highly purified human menopausal gonadotropin (HP-hMG), delivers 75 IU FSH and 75 IU LH activity. This product is a purified preparation of gonadotropins extracted from the urine of postmenopausal women. Three gonadotrophins were observed: FSH, LH and human chorionic gonadotrophin (HCG). The immunoactivity for HCG was three-fold higher than the immunoactivity for LH. In addition to gonadotrophins, a number of other proteins were detected. The amount of these impurities, as determined by reversed-phase high-performance liquid chromatography on a peak-area basis, is at least 30%. Therefore, it is concluded that this HMG preparation contains at most 70% gonadotrophins. Via a proteomics approach three major impurities were identified: leukocyte elastase inhibitor, protein C inhibitor, and zinc-alpha(2)-glycoprotein. Comparing batch-to-batch consistency using standard analytical techniques, as well as a novel in-vitro follicle bioassay, Oligosaccharide isoform profiling, immunoassay testing, size exclusion chromatography analysis and in-vitro bioassay testing confirm that the product  display a high degree of batch-to-batch consistency.

Recombinant human gonadotrophins (FSH, LH, hCG)

Following the development of highly purified urinary FSH, considerable improvements have facilitated both separation of FSH from human LH (hLH) and its production using recombinant technology. The structural complexity of human gonadotropins  and the need for post-translational modification of the molecule by protein folding and glycosylation, made functional protein production impossible in prokaryotes. Thus, a mammalian cell culture system was employed with functional molecules being produced in Chinese hamster ovary (CHO) cells. The world’s first recombinant hFSH (rhFSH; follitropin alfa) preparation for clinical use was produced by Serono Laboratories in 1988, and was licensed for marketing in the European Union as GONAL-f® in 1995. An rhFSH (follitropin beta; Puregon) product was also licensed by Organon Laboratories (now part of Schering Plough) in 1996.
The genes for the other gonadotropins have also been transfected into mammalian cell lines, and recombinant hLH (rhLH) and human chorionic gonadotropin (rhCG) are now commercially available.


Quantifying and standardizing gonadotropin content
Traditionally, quantification of hFSH, LH and hCG for clinical use has involved the use of invivo bioassays. For hFSH, a number of bioassays have been assessed for this purpose, but one of the most robust and specific remains the Steelman–Pohley in vivo assay, first developed in the 1950s. FSH activity is quantified by rat ovarian weight gain and FSH vials or ampoules are subsequently filled according to the desired bioactivity, measured in international units (IU). However, the assay has a number of limitations: it is time consuming, cumbersome, uses large numbers of rats (which is of ethical concern) and is limited in its precision—the European Pharmacopoeia defines an activity range (80–125% of the target value) within which an FSH batch is acceptable for clinical use.

Molecular modeling and the development of ‘designer’ gonadotropins
One approach for expanding the range of recombinant gonadotropins available for the treatment of subfertility is through protein engineering. Using today’s medications, daily injections of FSH are necessary for effective ovarian follicle stimulation. One scenario for the future is the development of new FSH molecules engineered to possess an extended half-life and duration of therapeutic action. Such molecules will enable the physician to provide single injections to drive follicle growth for up to a week in a controlled and predictable fashion.
One such long-acting protein, designated FSH– C-terminal peptide (FSH–CTP) was developed by Organon (now part of Schering Plough). FSH–CTP consists of the alfa subunit of rhFSH together with a hybrid beta subunit made up of the beta subunit of hFSH and the C-terminal part of the beta subunit of hCG. FSH–CTP has a longer half-life than standard rhFSH. A study in healthy female volunteers showed that a single dose of FSH–CTP induced multiple follicular growth accompanied by a dose-dependent rise in serum inhibin-B.  The first live birth resulting from a stimulation cycle with FSH-CTP was reported in 2003 and further studies have been carried out in subfertile patients undergoing ART and OI.

drugs4


Gonadotropin-releasing hormone agonist
Introduction

Control of gonadotropin secretion is exerted by hypothalamic release of GnRH, initially known as luteinizing hormone-releasing hormone (LHRH), but the lack of evidence for a specific FSH-releasing hormone (FSHRH) prompted a change in terminology. Gonadotropin-releasing hormone is produced and released from a group of loosely connected neurons located in the medial basal hypothalamus, primarily within the arcuate nucleus, and in the preoptic area of the ventral hypothalamus. It is synthesized in the cell body, transported along the axons to the synapse and released in a pulsatile fashion into the complex capillary net of the portal system of the pituitary gland.
Gonadotropin-releasing hormone was first isolated, characterized, and synthesized independently in 1971 by Andrew Schally and Roger Guillemin, who were subsequently awarded the Nobel Prize for their achievement. The structure of GnRH is common to all mammals, including humans, and its action is similar in both males and females. Gonadotropin-releasing hormone is a single-chain peptide comprising 10 amino acids with crucial functions at positions 1, 2, 3, 6, and 10. Position 6 is involved in enzymatic cleavage, positions 2 and 3 in gonadotropin release, and positions 1, 6, and 10 are important for the three-dimensional structure. 
In humans, the critical spectrum of pulsatile release frequencies ranges from the shortest interpulse frequency of approximately 71 minutes in the late follicular phase to an interval of 216 minutes in the late luteal phase.

GnRH agonists: Biosynthesis and mechanism of action
Native GnRH has a short plasma half-life and is rapidly inactivated by enzymatic cleavage. The initial concept was to create substances that prolong the stimulation of gonadotropin secretion. Analogs with longer half-life and higher receptor activities were created by a structural change at the position of enzymatic breakdown of GnRH.
The first major step in increasing the potency of GnRH was the substitution of glycine number 10 at the C-terminus. While 90% of the biologic activity is lost with splitting of the 10th glycine, it is predominantly restored with the attachment of NH2- ethylamide to the proline at position 9. The second major modification was the replacement of the glycine at position 6 by D-amino acids, which decreases enzymatic degradation . The combination of these two modifications was found to have synergistic biologic activity. Agonistic analogs with D-amino acids at position 6, and NH2-ethylamide substituting the Gly10-amide, are not only better protected against enzymatic degradation, but also exhibit a higher receptor binding affinity. The affinity could be further increased by introduction of larger, hydrophobic, and more lipophilicamino acids at position number 6. The increased lipophilicity of the agonist is associated with a prolonged half-life, which may be attributed to reduced renal excretion through increased plasma protein binding, or fat tissue storage of nonionized fat-soluble compounds.
Intranasal spray is extremely effective, but the bioavailability is only 3–5%, and the relatively fast elimination kinetics requires frequent dosing (2–6 times/day) to obtain continuous stimulation and down regulation.
For long-term treatment a depot formulation is available. The drug is formulated as controlled-release depot preparations with the active substance dissolved, or encapsulated, in biodegradable material. Intramuscular injections provide maintained therapeutic levels for 28–35 days. Thus, monthly injections are sufficient for maintaining down regulation.

Acute administration of GnRH agonistic analogs increases gonadotropin secretion (the flare-up effect) and usually requires 7–14 days to achieve a state of pituitary suppression. Prolonged administration of GnRH agonistic analogs leads to downregulation of GnRH receptors.

drugs11

drugs6

Side-effects
The main symptoms of low serum concentrations of estrogen are flushes, decreased libido, impotence, vaginal dryness, reduced breast size, and emotional instability.

drugs7

The structure of gonadotropin-releasing hormone (GnRH) and GnRH agonistists along with their different potencies.

Trade names, plasmatic half life, route of administration, and recommended dose for the clinically available gonadotropin-releasing hormone analogs (GnRH-a).

drugs8

Teratogenic effects
There does not appear to be an increased risk of birth defects, or pregnancy wastage in human pregnancies exposed to daily low-dose GnRH agonist therapy in the first weeks of gestation. From their toxicology studies in animals, no toxic effects were reported by the drug manufacturers.

Gonadotropin-releasing hormone antagonist: Mechanism of action

Antagonist analogs of GnRH have a direct inhibitory, reversible suppressive effect on gonadotropin secretion. Antagonistic molecules compete for and occupy pituitary GnRH receptors, thus competitively blocking the access of endogenous GnRH and precluding substantial receptor occupation and stimulation. Suppression attained by GnRH antagonists is immediate (no flare-up effect), and, as receptor loss does not occur, a constant supply of antagonists to the gonadotroph is required to ensure that all GnRH receptors are continuously occupied. Consequently, compared with agonistic analogs, a higher dose range of antagonists is required for effective pituitary suppression.

Synthesis of GnRH antagonists
The first generation of antagonistic analogs were hydrophilic, and contained replacements for His at position 2 and for Trp at position 3. Inhibitory activity increased after incorporation of a D-amino acid at position 6. However, histamine release also increased, resulting in anaphylactic reactions which prevented their clinical use. In third-generation antagonistic analogs, the undesirable risk of anaphylaxis and edema was eliminated.

drugs9
The structure of gonadotropin-releasing hormone (GnRH) and GnRH antagonists

Safety and tolerability studies
The introduction of GnRH antagonists in clinical use was delayed owing to the property of the first generation of antagonists to induce systemic histamine release and a subsequent general edematogenic state. Effects of GnRH antagonists on the ovary, oocytes, granulosa cells, endometrium, and the embryo in relation to fertility and implantation rates are being investigated. Direct effects of GnRH antagonists on human ovarian steroidogenesis in vitro have not been demonstrated. In preliminary in vitro and animal studies, recent data revealed that some adverse effects on oocyte maturation and on preimplantation development of embryos may be inflicted by GnRH antagonists through inhibition of GnRH receptors in these structures.

Comparing mechanisms of action of gonadotropin-releasing hormone (GnRH) agonists and antagonists.

drugs10

Gonadotropins Trade Name

Human Menopausal Gonadotropins
Pergonal
Menogon
Merional

Purified hMG

Menopur
Repronex

Purified FSH
Metrodin
Bravelle
Fertinex

GnRH agonists trade name
Lupron
Luprolide
Decapeptyl
Nafarelin
Buserelin
Zoladex
Synarel
Enantone
Procin

GnRH antagonists trade name

Antagon,
Ganirelix,
Cetrotide
Orgalutran
Cetrorelix

Recombinant FSH trade name
Gonal-f
Gonal-f REF
Gonal-f REF Pen
Puregon
Puregon Pen

Recombinant  LH trade name

Luveris

Clomid (Clomiphene Citrate) Trade Names

Ardomon, Clom 50 (o.c),  Clomiphenecitrate, Clomifen, C.-ratioph. (o.c), Clomiphen Citrate,  Clomiphen-Merck, Clomipheni citras, Clomivid, Clostilbegyt, Clostilbegyt, Clostilbegyt (o.0 G, Dufine,  Dyneric, Gravosan, Indovar, Klomifen,  Kyliformon, Pergotime , Omifin, Pioner, Prolifen, Serofene,  Serophene, Serpafar,  Tokormon

Urinary Human chorionic gonadotropin (HCG) trade name
Generic Name: human chorionic gonadotropin (HCG)
Brand names: Novarel, Pregnyl, Profasi, Chorex, Gonic, HCG, Chorigon, Choron-10, Profasi HP

Recombinant Human Chorionic Gonsdotropin trade name
Ovidrel