Pharmacology of Human Reproduction PDF

Lecture 3 - 16/10/2023 PHARMACOLOGY OF HUMAN REPRODUCTION The endocrine system is one of the two systems responsible for communication and integration of specific inputs between tissues-cells-organs; the other is the nervous system. Endocrine communication is achieved...

Lecture 3 - 16/10/2023 PHARMACOLOGY OF HUMAN REPRODUCTION The endocrine system is one of the two systems responsible for communication and integration of specific inputs between tissues-cells-organs; the other is the nervous system. Endocrine communication is achieved by chemical messengers, the hormones. Hormones can be divided into two different classes: Lipid-soluble molecules → these hormones interact with specific receptors located inside the cells and act as transcription factors. Water-soluble molecules → they can’t diffuse into the cells but interact with specific molecules on the membrane, they produce their effects by the activation of G-proteins, channels… Hormones are chemical messengers that circulate in the body fluids and their action is far from the site of production of the hormone itself: hormones are released into the blood circulation and by using vessels they are distributed into the human body. Obviously, only the cells that have the receptors for the hormones can respond to it. The major functions of hormones include the regulation of energy storage, production and utilization, the adaptation to new environments or conditions of stress, the facilitation of growth and development and the maturation and function of the reproductive system. There are three types of signaling: Endocrine → the hormone is released in the blood circulation and, through the blood vessels, it exerts its action far from the site of production so the cell that produce the hormone is far from the target cell. Autocrine → the chemical messenger modulates the cell that produced the hormone itself; for example, cytokines work as autocrine signaling. Paracrine → the cell target is near the cell that produce the hormone. Regarding the signaling of hormones, there are two different classes: Hormones that act via nuclear receptors, that modulate transcription in target cells; steroid hormones, thyroid hormones, vitamin D are lipid soluble. Hormones that act via membrane receptors, that exert very rapid effects on signal transduction pathway; this type of hormones are water-soluble. The HPA axis (hypothalamic pituitary endocrine axis) is made by the hypothalamus and the pituitary. Hypothalamus is a brain region, and the hypothalamic neurons produce some releasing/inhibiting factors that are transported through the median eminence at the pituitary level. The releasing/inhibiting factors induce the release/inhibition of specific hormones from the pituitary gland. The pituitary gland can be divided into: Adenohypophysis, or anterior pituitary → it produces 1  hormones like ACTH, FSH, LH, GH, PRL. Neurohypophysis, or posterior pituitary → it doesn’t produce hormones itself but receives some factors produced by the hypothalamus and then it releases them in the blood circulation, vasopressin e oxytocin. Adenohypophysis can produce hormones after receiving the releasing factors from the hypothalamus while neurohypophysis is a brain region that receives directly inputs from neurons at hypothalamic level, leading to the production of oxytocin and vasopressin. The hormones produced by anterior pituitary are: POMC (proopiomelanocortin) -derived hormones → include adeno-corticotropic hormone (ACTH) and melanocyte stimulating hormone (⍺-MSH). Somatotropin hormones → Growth hormone (GH) and Prolactin (PRL); in humans, the somatotropin family also includes placental lactogens. Glycoprotein-based hormones → TSH, LH, FSH, hCG. Hormones produced at hypothalamic level are released in the posterior pituitary, also known as neurohypophysis, that is a nervous region that contains the endings of neurons located into the hypothalamus. These hormones are: Vasopressin, involved in the regulation of water homeostasis. Oxytocin, involved in labor and milk ejection. The production of hormones is under the control of the hypothalamus; for example, GH receives two different types of modulation: GH is produced by the anterior pituitary and it is modulated by the GHRH secreted by the hypothalamus, able to increase the release of GH. On the contrary, somatostatin can reduce the releasing of GH by the anterior pituitary. Other examples: Corticotropin, that induces the release of cortisol by the adrenal glands, is stimulated by corticotropin releasing hormone. FSH and LH are under the control of GnRH, secreted by the hypothalamus. 2  GROWTH HORMONE There are some kinds of cells that produce GH and PRL and this production is under the control of the hypothalamus. The endocrine regulation of GH is stimulated by GH releasing hormone, but its production is negatively regulated by somatostatin (SST); also, Ghrelin, produced by the stomach, can induce the production of GH. The production of GH is modulated during the day, in particular, the secretion is very high during infancy, it peaks during puberty and decreases in adulthood. GH is produced in a pulsatile manner during the day and night: the maximum of production, the peak, is during night, stimulated by GHRH and Ghrelin; the inhibition is by somatostatin, GH itself and IGF-1. Once released into the circulation, GH binds and activates the cell-surface GHR. It is a key regulator of insulin- like growth factor 1 (IGF1) which is secreted from target tissues, particularly by the liver. Increased serum GH and IGF1 produce feedback loops that lead to inhibition of GH secretion from the pituitary. The effects of GH are to stimulate growth, in particular to promote the postnatal longitudinal growth of the bones. It increases glycolysis and body fat, important to maintain muscle mass and its strength, in the immune system, cardiovascular and for the well-being of the nervous system. From a pathological point of view, the conditions of abnormality of GH are: GH hyper-secretion → abnormal levels of GH lead to gigantism in childhood and acromegaly (longer legs and arms) in adulthood because GH promotes the longitudinal growth. Congenital disruption of GH signaling → deficiency in GH or alteration in signaling of GH receptors, leading to short stature or some specific and rare syndromes. GH receptors GH receptors are very similar to cytokine receptors, characterized by the Janus Kinase 2 (JAK2): in fact, these receptors lack intrinsic kinase activity and require the recruitment of non-receptor tyrosine kinase, JAK2. When GH interacts with GH receptors, activates the JAK, which phosphorylates some downstream targets like STAT5, there is the signal transduction, STAT5 dimerizes and enters the nucleus and modulates the transcription of specific genes. Genes that are modulated by GH are involved in the growth, metabolism, and sex dimorphisms. GH, besides acting on the liver, bones, adipocytes, and muscles, can interact by inducing another second messenger, which is insulin growth factor 1 (IGF-1). 3 Two hypothalamic factors, GHRH and SST, stimulate or inhibit the release of GH from the pituitary, respectively. IGF-1, a product of GH action on peripheral tissues, causes negative feedback inhibition of GH release by acting at the hypothalamus and the pituitary. The actions of GH can be direct or indirect (mediated by IGF-1). Ghrelin can stimulate the release of GH and it has this important effect on GH because with the intake of food there is the stimulation of the secretion of GH. When there are high levels of IGF-1, there is a mechanism of negative feedback in the production of GH. Pathophysiology of GH In case of an excess of production of GH, the hypotheses can be the presence of some adenomas that over secrete GH; in condition of deficiency, children will have short stature while adults will have decreased muscle mass, decreased bone density, impaired psychological functions, and increased mortality from cardiovascular causes. From a pharmacological point of view, if the hyper-secretion of GH is caused by tumors, the first step is to remove by surgery the adenomas; to reduce the effects of the excess production of GH, it’s possible to use some somatostatin analogues (block the production of GH) or GH antagonist (block the downstream effects produced by the activation of GH receptors). One of the most important drugs is Octeotride: it is a drug administered subcutaneously and it is a somatostatin analogue, so mimics what SST does (blocks the release of GH). It is administered three times a day, its effect is very rapid (within 30 minutes) but there are some side effects, such as diarrhea, nausea, and abdominal pain. Octeotride is 40 times more potent than somatostatin itself and it is characterized by a long half-life; the goal is to reduce GH levels. It binds preferentially to receptors on GH secreting tumors/decreasing tumor size. Another approach can be to cancel the effect of hyper stimulation of GH receptors using GH antagonist, which are drugs that block GH receptors. Pegvisomant is an antagonist approved for the treatment of acromegaly; it binds to the GH receptors and doesn’t activate JAK-STAT signaling or stimulate IGF-1 secretion. It is administered subcutaneously. In case of deficiency of GH, the approaches can be: Administration of GH itself. Use of IGF-1 since the effects of GH are mediated by the increase of IGF-1. Use of GH releasing hormone to facilitate the production of GH by the pituitary. These different approaches depend on the cause of GH deficiency. The administration of GH is a replacement therapy when the child is not able to produce GH itself and has been diagnosed with GH deficiency; in the past the hormone came from the pituitary of cadavers but today is produced thanks to the recombinant DNA technology. It is a very important therapy because requires almost six months before seeing the beneficial effects (e.i. increase of the stature); it is administered subcutaneously, with a bioavailability of 70%. Some side effects can 4 be allergic reactions. Another approach is to inject into the patient Insulin Growth Factor 1 (IGF-1) together with a drug called mecasermin. This drug can be used when the problem of deficiency is related to the fact that the receptors don’t work but the levels of GH are high. In fact, IGF-1 is involved in some of the effects mediated by GH, and it has been seen that, by administering IGF-1, it is possible to obtain the functioning of the receptors. Mecasermin is FDA-approved for patients with deficiency of GH due to mutation in GH receptors or in alteration of GH receptor signaling; it is administered subcutaneously. The side effects of mecasermin are hypoglycemia and lipohypertrophy. The last approach is the administration of GH releasing hormones in order to induce the release of GH itself, mimicking what the hypothalamus does. The synthetic peptide is Tesamorelin, it is resistant to the degradation of dipeptidyl peptidase and therefore has a prolonged duration of action. Tesamorelin can increase the levels of GH and IGF-1 but its clinical effects are primarily to reduce visceral fat accumulation, with minimal effects on insulin resistance. Summary of GH-related drugs Lecture 4 - 17/10/2023 Growth hormone overview We looked at anterior pituitary activity and what we saw is that there are different types of cells and we talked about cells that are producing growth hormone and prolactin. So basically, from a pathological point of view, we can have two different conditions. One is characterized by excessive secretion of growth hormone, and we have deficiency in the children. So, we can use two different compounds in addition to GH releasing hormone. We can use octreotide which is a somatostatin analogue. Or we can inhibit growth hormone receptor activity by using growth hormone antagonist, for example, pegvisomant. The opposite pathological condition is characterized by short stature or a specific syndrome in which we can have a deficiency of GH or the signaling associated with GH receptor doesn’t work. So, we can also do a replacement therapy with analogues of growth hormone, for example, somatotropin. Or we can bypass growth hormone receptor activation if there are mutations in the GH receptor, by injecting these patients with mecasermin which is recombinant human IGF1. 5 PROLACTIN —> production of milk OXYTOCIN —> milk breakdown PROLACTIN Next, we have prolactin. So, prolactin is another hormone which is very important during, after, labor to produce milk and to induce milk production. This hormone is a protein, and its function is specifically characterized in females but there is also in males although its role has not been medically clarified. First of all, what is the role of prolactin? It is to produce milk. However, prolactin also acts as a cytokine, so it is very important to modulate the immune system. And probably being a growth factor, it is also involved in the maturation of blood cells, so it influences hematopoiesis. What is the mechanism of regulation of prolactin secretion? The mechanism is a little bit complex since prolactin is produced by lactotrophs cells which are located in the anterior pituitary. And these lactotrophs cells are indeed regulated by some specific neurons coming from hypothalamus, which are called TIDA neurons. Tuberoinfundibular neurons (TIDA) are dopaminergic neurons. So, you have to imagine that we can have these dopaminergic neurons from specific nuclei (arcuate) of the hypothalamus in particular this this region (anterior pituitary). And these dopaminergic neurons regulate lactotrophs cells whose role is to produce prolactin. So, as you can see here is an overview of the mechanism of regulation of prolactin. Prolactin is produced by anterior pituitary and like growth hormone is a growth factor and is under the influence of TRH. As you can see here, TRH from hypothalamus induces an increase of prolactin, while dopamine exerts a negative regulation. And the last thing is that prolactin exerts its effect essentially in breast by stimulating the production of milk and milk ejection, but also in other tissues and of course in females. What is the primary stimulus to produce prolactin? It is the survival of the baby. So, what we have to remember looking at the slides is this overview, the mechanism of regulation. So, prolactin is produced by lactotrophs and these lactotroph cells are under the inhibitory control of the dopaminergic neurons from hypothalamus and these neurons are called TIDA. What happens basically is that these neurons TIDA are here in the hypothalamus. This is hypothalamus and this is anterior pituitary (she shows on photo). Here in the hypothalamus we have these neurons, as you can see here. They release dopamine where we have median eminence. So, at this level dopamine reaches the anterior pituitary and interacts with the specific receptors that are located on lactotrophs cells. And these receptors are D2 dopamine receptors. So, what happens basically is that the activation of dopaminergic neurons exerts inhibition of prolactin. Prolactin is not produced. Why? Because of dopamine release from the TIDA neurons and dopamine reaches lactotrophs cells. Dopaminergic receptors are generally classified into two different classes, and they are called D1 light receptor and D2 light receptor. These are two different families of dopaminergic receptors. D1 light receptors are GPCR 6 dopamine is released from hypothalamic neurons, interacts with D2 dopamine receptors DA receptors D1 D2 Gs Gi lack of production of prolactin coupled with GS, G stimulatory protein. While D2 light is a certain family of receptors that are GPCR receptors coupled with GI, G inhibitory protein. So, if I want to see that lactotrophs cells express on their surface these receptors which are coupled with GI inhibitory proteins, dopamine will interact with these dopamine receptors and will induce a mechanism of inhibition. So, lactotrophs cells are not able to produce prolactin. So, dopaminergic activity of these TIDA neurons exerts the mechanism of inhibition. Dopamine, by interacting with these inhibitory receptors (D2) produces the lack of prolactin secretion. This stimulus of suckling is very complex stimulus. However, this stimulus induces the inactivation of these dopaminergic neurons. So, when TIDA neurons are in some way deactivated we have the lack of this inhibitory control and prolactin is produced by lactotrophs. The system is a little bit complicated since, as you can see here, there is also a short loop feedback exerted by prolactin because prolactin stimulates, for example, dopaminergic neurons. When there is a production of prolactin, it reduces its further production by stimulating the dopaminergic neurons. Another important aspect of this circuit is that prolactin also inhibits gonadotropin releasing. This is the reason for which hyperprolactinemia female patients can have irregular menses with amenorrhea or oligomenorrhea. These systems are strongly interrelated, and we can have hyperprolactinemia with irregular menses. It is very important because it interferes with the fertility and pregnancy success rates. As I told you before, prolactin functions are limited primarily to the mammary gland. However, prolactin receptors are distributed also in other organs and also in men. The physiological effect of prolactin on other sites has not been characterized, particularly in males. that’s why it’s less liely for lactating mothers to +casein + lactalbumin = milk production be pregnant and also why conditions of Pathophysiology of prolactin GnRH = anovulation hyperprolactinemia are related to infertilty Now we will discuss the pathophysiology of prolactin in a particular condition, which can be very common, and is hyperprolactinemia. Hyperprolactinemia is a very common endocrine abnormality, and the causes can be different. For example, we can have some hypothalamic or pituitary adenomas and we can have some pharmacological treatment. What are the symptoms of hyperprolactinemia in women? We can have galactorrhea, amenorrhea, and infertility. There are some important symptoms also in men, like loss of libido, erectile dysfunction, and infertility. And also, in men we can have gynecomastia which is the enlargement of breast tissue in men. This condition needs to be treated from professors’ point of view. Causes can be different from tumor to some dysfunction of thyroid gland or specific pharmacological treatment like for example antipsychotics. And the symptoms are different of course in males and females. In the right picture you can see how prolactin is correlated with infertility. As you can see here, prolactin production is controlled by TRH and dopamine, as I told you before, but there are some other mechanisms involved. For example, the main neurogenic stimulus to produce prolactin is represented by breastfeeding, but also estrogen during the pregnancy can increase prolactin production. Also, some specific conditions like polycystic ovarian syndrome or pituitary tumors like 7  adenomas can induce the production of prolactin. TRH also stimulates prolactin production and medication as well. Specific medication like for example, neuroleptics or antipsychotics can induce an abnormal prolactin production. That is because antipsychotics act as antagonists of D2 receptors. So, if I block the activity of D2 dopamine receptor that exerts inhibitory control on lactotroph cells, these cells then produce prolactin. So, as you can see here, a lot of medications can induce a condition of hyperprolactinemia. Antipsychotics are antagonists of D2 receptors. And D2 receptor is a GPCR with the GI inhibitory protein. So, when dopamine interacts with this receptor it inhibits the production of prolactin. In some way if I use an antagonist, I'm having a situation in which I have no stimulation of dopaminergic neurons. So, antipsychotics by blocking D2 receptor on lactotrophs cells in some way impair the regulation of lactotrophs cells themselves. This way we can have an abnormal, an ectopic production of dopamine. Why is it important for us? This condition of hyperprolactinemia? Because it interferes with fertility. If I receive people with problems of fertility, the first question, the first test that I have to do is to test the level of prolactin, because prolactin is a quite common endocrine dysfunction. So, if I have a condition characterized by increased levels of prolactin, I have to start a pharmacological treatment. If this condition is caused by tumors, I have to remove by surgery the tumor itself. Otherwise, I can start the treatment with specific drugs. In this case, specific drugs with the dopamine D2 receptor agonists like bromocriptine, cabergoline, and quinagolide. So, again, as you can see here this is the mechanism of dopamine exerting inhibition control of prolactin production (photo above). So, if I block in some way dopamine like with antipsychotic drugs, the final effect is an increase of prolactin. So, dopamine agonists are the golden standard for the treatment of hyperprolactinemia. Dopamine agonists bind D2 dopamine receptors located on lactotrophs cells and activate them. And since D2 dopamine receptors are GPCR with the GI inhibitory protein, the final effect of this activation is the reduced production of prolactin. They effectively reduce prolactin levels, thereby relieving the inhibitory effect of hyperprolactinemia on ovulation and permitting most patients with prolactinomas to become pregnant. Bromocriptine, cabergoline, and quinagolide effectively reduce prolactin levels and the effect of prolactin on Treatement:: - surgery ovulation, and this is a very important treatment to restore fertility of the patients and to restore normal menses. pamine receptor agonist: high level of dopamin have been asociated with psycosis, allucination Of course, with increasing doses of dopamine agonists we can have some important side effects at the CNS level, central nervous system level. And we can have, of course, psychosis, hallucinations, nightmares, insomnia etc. High levels of dopamine can induce some psychotic features. Bromocriptine is a good derivative. It is a D2 agonist and D1 partial agonist. So, we can have some side effects due to the effect on D1 receptor. And we can have some nausea, vomiting, and vomiting probably is induced by stimulation of the brain stem vomiting center. Cabergoline is another good derivative, and it is the first line agent to treat prolactinomas because this drug shows very high affinity for D2 receptors and is characterized by less frequent side effects. Pharmacological approach is based on D2 agonist So essentially, medications used to treat hyperprolactinemia are represented by these two different drugs which are bromocriptine and cabergoline; both D2 dopamine receptor agonists although in some circumstances they can also have higher affinity for other receptors like D1. However, they are very important to restore fertility in female in order to obtain regular menses, so the final effect is that women are not affected. dopamine agonist are also used to prevent ovarian hyper stimulation 8  agonist cotinuous treatment —> down-regulation of receptor antagonist conintinuous tratement —> up-regulation of receptor GONADOTROPINS AND GnRH Next, we will discuss gonadotropins, which are essentially LH, FSH and hCG. Gonadotropins are relative compounds. These are of course agonists, but we hypothalamic releasing factor can have also antagonists. They are called gonadotropins since their action has been clarified at the beginning of gonads. hCG is produced by placenta. This is the mechanism of regulation of gonadotropins. They are produced again by anterior pituitary and regulated by the single hypothalamic factor called gonadotropin releasing hormone (GnRH), which controls the synthesis and the release of both the gonadotropins LH and FSH, both in males and females. What LH and FSH do is stimulate the gonads to produce sexual hormones, steroids and also inhibin, which is a hormone that specifically inhibits FSH. As you can see here, we have specific mechanisms of regulation. So, the stimulation in the production of gonadotropins is represented by GnRH produced at hypothalamic level. And this is also a mechanism of feedback inhibition produced by sex hormones themselves. When there are high level of estrogens or testosterone, these sex hormones induce different feedback effects (feedback inhibition) at the hypothalamic level. So, there is no release of GnRH. feedback mechanisms dependent on the concentration of sex steroids Gonadotropin’s structure Looking at the receptors, we can see that TSH is really similar to thyoid one Gonadotropins share a basic structure. They are heterodimeric compounds (glycosylated heterodimers) characterized by an alpha subunit and beta subunit. So, as you can see here, there is an important picture we have. LH is composed by this structure (alpha subunit, LH beta subunit and one N molecule). FSH is different because it has two N molecules. As you can see here TSH is very similar in structure to LH. This is why its dysfunction at thyroid level can impair fertility. It can alter menses regularity for example. So, hormone dysfunction in some way can alter the menses cycle and fertility. And the reason is that, as you can see here, if I look at the structure, the organization of LH, for example, I see that this structure is very, very similar to TSH. hCG has an etension of C-terminal that leads to a longer t1/2 So, what happens when, for example, I have a condition characterized by low levels of thyroid hormones? When we have low levels of thyroid hormones TSH is raised after. Due to that, we can have some alterations in the ratio between TSH, FSH, and LH and this leads to abnormal menses alterations in the menstrual cycle. This is the reason I often say that this pharmacology is very complex because there is not a single hormone and a single effect. But these mechanisms, these disciplines are strongly interrelated with each other. It is difficult in some circumstances to predict the event only based on the drug administered. hCG, as you can see, is very similar to the other compounds. But this structure is a little bit more complex than in LH. For this reason, hCG has a longer half-life than LH. And this is why during, for example, in vitro fertilization protocols, I administer hCG in the place of LH. In order to understand this point, you have to consider that in in vitro fertilization protocols, I have to induce the maturation of the follicle and the rupture of the follicle. And to induce this point, I have to administer LH because the LH surge is important for ovulation. I have to inject the LH. 9 In the clinical practice I administered not LH but hCG. Why? Because hCG is characterized by a longer half-life and it has a longer half-life because the structure is more complex. So, instead of LH I generally in the clinical practice inject hCG because it has a longer half-life. Physiology of gonadotropins So, what are the effects of gonadotropins in males? As you can see here, we can have both LH and FSH. LH stimulates Leydig cells to produce testosterone from cholesterol, while FSH stimulates the Sertoli cells to increase nutrients intake of amino acids in order to favor the maturation of sperm. What happens in women? So, in this case the situation is very complex because FSH stimulates the growth of developing ovarian follicles and at the same time induces the expression of a receptor for LH on theca and granulosa cells. FSH also regulates the expression of aromatase in granulosa cells, which is the enzyme necessary to produce estradiol. LH exerts its effect on theca cells and stimulates de novo synthesis of androstenedione, which is an important precursor of estrogen in women (premenopausal women). LH in particular is necessary for the rupture of dominant follicle during ovulation and for the synthesis of progesterone by the corpus luteum. This is an example of what happens during menstrual cycle, and you can see here the cycle is seen from different points of view. What happens in the anterior pituitary is depicted here (A) and also what happens in the ovary (B). Here we have what happens in terms of ovarian hormones (C), estrogen and progesterone. And here are events that happen in the endometrium of uterus (D). The time point zero is the first day of menstruation, of the bleeding. During this phase FSH and LH tend to increase but there is only a slight increase. During this time, in particular, FSH induces the increase of follicles then there is the maturation of a single follicle. And when that happens the LH surge occurs, there is a rapid increase of LH. We also have an increase of FSH, and we have ovulation with the rupture of the follicle and the release of the oocyte. What happens at the level of ovarian hormones? In this case we have the estrogen rise and there is the proliferation of endometrium. If a pregnancy does not occur, we have the degeneration of corpus luteum okay and degeneration of the endometrium which was induced to proliferate by estrogen. What happens in terms of different days of menstrual cycle? In the ovarian cycle we can define specific phases. As you can see here, we have the follicular phase which spans from the day 0 to 13. Then we have the ovulation around the day 14. And then we have luteal phase from day 15 to the end of the cycle, which culminates with the menstruation and the bleeding. Then the new ovarian cycle can begin. 10  not stimulated GHRH stimulated if I want to mimic this pulsatile secretion I need to administer 3/4 times a day —> production of FSH and LH, which increase if I use a continuous stimulation with a drug that has longer half-life, i have this condition: continuous graph —> downregulation FSH and LH decrease time receptors are exposed to a conunuous stimualtion from GHRH analogs —> downregulation 7-10 days FSH and LH, then a drop in their concenetration Gonadotropin releasing hormone (GnRH) As I told you before, the main trick when it comes to synthesis and the release of gonadotropins is represented by gonadotropin releasing hormone (GnRH). GnRH is hypothalamic peptide (decapeptide) produced by hypothalamus. GnRH is generally characterized by a pulsatile secretion and GnRH is controlled by hypothalamic neural pulse generator (primarily in the arcuate nucleus) which is located in the hypothalamus, and it controls the frequency and amplitude of GnRH release. Before puberty there is an inhibitory control of this circuit. But during puberty we have a strong increase of GnRH patterns. And this pattern lasts to adulthood. As puberty progresses, GnRH pulses increase further in amplitude and frequency until the normal adult pattern is established. You have to remember that the release of GnRH is pulsatile. birth & infancy —> inhibition of master block before puberty —> block removed Now we will talk about gonadotropin receptors. Gonadotropin receptors are GPCR coupled receptors. And in fact, the hormones are peptides. For hormones which are glycoproteins or peptides we can find receptors located on the membrane. Since these compounds are water soluble, they cannot diffuse into the cells. Due to that, receptors will be necessarily localized on the surface of the cell, and they are GPCR. As I told you before, in physiological condition the production of FSH and LH is regulated by the GnRH. GnRH is released in a pulsatile manner, which means that it is up and down, up and down during the day and during the cycle. We can have a lot of compounds. For example, we can have drugs that act as synthetic agonist analogs of GnRH. Leuprolide, for example, is GnRH synthetic analog. Analogs can have a different effect depending on the regimen of the administration. What happens if I treat a patient with a gonadotropin releasing hormone synthetic analogue by mimicking what happens at physiological level? So, if I mimic the release in pulsatile manner? If I use this compound, for example, to treat a condition characterized by the lack of gonadotropins and simulate what happens at the physiological level, in order to induce the generation of gonadotropins. What happens if I treat the patient with the synthetic analog in a continuous manner, not in the pulsatile manner? We have negative feedback. Negative feedback, in particular we can have the downregulation. What happens following the continuous activation of gonadotropin releasing hormone receptors is that these receptors undergo in some way, downregulation. So, the final effect is to reduce the production of gonadotropins. So, if I use these compounds mimicking the pulsatile manner of releasing, I can increase the production of FSH and LH. But when I use these compounds in a continuous manner, I can have downregulation of gonadotropin releasing hormone receptors and this leads to low levels of FSH and LH. So, essentially, we can say that the GnRH agonists can induce biphasic response if I use this compound by continuous infusion (administration). At first, I can have an increase in FSH and LH, but when receptors undergo downregulation, I can have a drop in the levels of FSH and LH. So, when I use GnRH agonists in a continuous treatment, what happens? I use gonadotropin releasing hormone agonist by continuous infusion in the first 3-4 days. During this time, I have an increase in FSH and LH, and this effect is called flare up because these agonists stimulate the receptor. The final effect is an 11  increase in FSH and LH. Then the mechanism of adaptation occurs. Now, we have the downregulation of gonadotropin releasing hormone receptors and the effect is the reduction of FSH and LH. So, we can have in the beginning flare effect due to the stimulation of gonadotropin releasing hormone receptor, then we have mechanism of adaptation and downregulation of the receptor. Why do we have downregulation of GnRH receptor? Because the receptor is used to see (interact) with the hormone released in a certain manner (pulsatile). But if the receptor is continuously stimulated then it is going to become downregulated. So, what is the effect of GnRH agonist? If we have the administration in a continuous way, we can have at the beginning a flare effect which leads to an increase in FSH and LH, then an adaptation without regulation of the receptors. Downregulation of the receptor leads to a reduction in the secretion of gonadotropins. On the other hand, if the agonist is administered in a pulsatile manner, so, if we have pulsatile intravenous administration every 4 hours, we are mimicking what happens at physiological level. In this way I can stimulate the FSH and LH secretion. How are these drugs (synthetic GnRH agonists) used? For example, they are used to treat precocious puberty, to suppress conditions which are very sensitive to estrogens like endometriosis, uterine fibroids, and other conditions. What are the adverse effects of these gonadotropin releasing hormone agonist? The long-acting agonists are generally well tolerated. We can have hot flashes when we stimulate the release of estrogens, or we can have some impairments in the vaginal mucosa and also erectile dysfunction. These are some examples of specific drugs. For example, leuprolide, which is a synthetic peptide with long-acting GnRH agonist activity. We can use this drug in children with precocious puberty and also in men with advanced prostate cancer, because this type of cancer is very sensitive to androgens. So, I can reduce the androgen production by regulating the GnRH receptor and the same happens in children with precocious puberty. This is the definition of precocious puberty which is the initiation of sexual maturation before age of 8 in girls or age of 9 in boys. We can use GnRH agonists with intent to treat such patients with continuous administration in order to reduce the production of gonadotropins. Also, these compounds are used during the in vitro fertilization protocols and to treat other conditions, like infertility in which we can have an impaired synthesis or secretion of gonadotropins (hypogonadotropic hypogonadism). In this case, these drugs are used in a pulsatile manner in order to mimic what happens physiologically during the menstrual cycle. intermitting dosing can be used to treat the late puberty or when there is impaired synthesis of proteins So here to sum up what we have said so far. When we talk about hypothalamic hormones and related drugs, we can GnRH-related preparations, like leuprolide, gonadorelin, nafarelin. The mechanism of action is to interact with the gonadotropin releasing hormone receptor. Depending on the type of the regimen of administration if it is continuous or pulsatile, I can have different effects. So, if I want to inhibit the gonadotropin release, I have to administer in a continuous way compounds characterized by long-acting activity and that have a longer half-life. If I want to stimulate the secretion of gonadotropins, I have to mimic the pulsatile manner and compounds should be administered every 1-4 hours in order to mimic the up and down manner of release of the hormone during the physiological condition. We can also have gonadotropin releasing hormone antagonists. These compounds exert their effect by interacting with the GnRH receptor and blocking it. We can have different compounds such as cetrorelix and ganirelix. Due to these drugs, there is no production of gonadotropins. 12 effect of antagoonist is immediate, in IVF we can have different protocols  GnRH agonist pulsatile dose continuous dosing treatement: compound that induce - precocious puberty downregulation of this receptor - endometriosis - delayed puberty - breast cancer - anovulatory infertilty - prostate cancer +IVF protocols What is the different effect of GnRH agonist or antagonist injected continuously? So, if I want to suppress immediately the release gonadotropins, I preferentially use gonadotropin releasing hormone antagonist because the antagonist blocks the receptor immediately. And, the final effect is to reduce gonadotropin production immediately. While GnRH agonist requires time in order to induce the downregulation of gonadotropin releasing hormone receptor. So, at the beginning you can experience a flare effect with the increase of FSH and LH and only later on you will experience the reduction in gonadotropin level. Different compounds are used during in vitro fertilization protocols in order to, for example, prevent premature ovulation (ganirelix, cetrorelix). And this is very important because during in vitro fertilization protocols, you can use both gonadotropin releasing hormone agonist or antagonist in order to prevent premature ovulation. There are IVF protocols based on gonadotropin releasing hormone agonist or antagonist. At the moment we can think that this is a nonsense, that it is impossible that an agonist or an antagonist exert the same effect. However, the effect of the agonist depends on the regimen of administration. So, if GnRH agonist is administered in the continuous way, I can obtain a downregulation of the receptor, and this leads to a reduction in the secretion of gonadotropins. hypersensivity reactions are the main side effect Some of these compounds are also used in the prostate cancer and degarelix is one of the most important drugs used in the treatment of advanced prostate cancer. The final effect is to reduce testosterone levels. As I told you before, that prostate cancer is a type of cancer that is sensitive to testosterone. So, by using the GnRH antagonist we can have a reduction in testosterone levels. However, we can have some important side effects like hot flashes, weight gain, and some side effects in the liver. Since all these side effects depend on low levels of testosterone, this condition is quite difficult to accept by men because there are a lot of side effects related to sexual activity, for example, and also to muscle mass. That is because testosterone is very important to increase and to sustain the muscle mass and the strength of the muscle. Another side effect is the decrease of bone mineral density, because again, testosterone is very important to maintain bone mineral content. By using these compounds which are approved for the treatment of advanced prostate cancer, I mimic a condition for medical castration. Natural and recombinant gonadotropins When we talk about gonadotropins, we know they are regulated by GnRH produced at hypothalamic level. Now, we have compounds that act both as agonists and antagonists of GnRH receptor. Then we have gonadotropins. Of course, I can also administer gonadotropins like LH and FSH. These compounds are classically purified from some chemical reactions by using recombinant DNA technology and exhibit less batch-batch variation. This technology is being used to produce forms of gonadotropins with increased half-lives or higher clinical efficacy. In the past, they were prepared from human urine and included CG, obtained from urine of pregnant women and menotropins, obtained from urine of postmenopausal women. Since they had low purity, they were administered intramuscularly to decrease hypersensitivity reactions. Today they are prepared by using DNA recombinant technology and this is very important because this type of production is very consistent while the preparation from human urine was characterized by a lot of side effects, in particular hypersensitivity reactions. They are all compounds administered parenterally. There are different compounds. The names are not important. For example, we have menotropins which are a mix of FSH and LH purified from the urine of postmenopausal women. Today we can have FSH and its analogues like urofollitropin, follitropin alpha and beta, and so on. Or we can administer LH and its analogs. As I told you before, LH is not used because it is characterized by a short half-life. 13 Instead, we used hCG which has a structure that is nearly identical to LH and mediates its effects through activation of LH receptors. hCG purified from human urine or recombinant hCG is used commonly for an LH activity. Lutropin, a recombinant form of human LH, is also available. For example, FSH is used to induce ovarian stimulation or during in vitro fertilization protocols. LH is not used, but hCG because it is characterized by the long half-life. Natural and recombinant gonadotropins are used, also, in male infertility. For example, in conditions characterized by gonadotropin deficiency. We can have some important side effects like gynecomastia. They are also used in situations like cryptorchidism. However, often patients first undergo surgery and then later IVF protocol. These compounds are frequently used. They can be used in ovarian stimulation, as you can see here. We can the natural cycle and, of course, there is no medication. There are different types of ovarian stimulation for in vitro fertilization protocols. We can use this modified natural cycle so we can administer, for example, hCG only. But we can also have other protocols like the conventional protocol characterized by the administration of gonadotropin releasing hormone agonist or antagonist, and then FSH to induce the ovarian stimulation. We will see these different protocols one by one. This is an example of in vitro fertilization protocols. As you can see here, we can have two different protocols based on agonist long protocol or antagonist-based protocols. 14  Lecture 5 - 19/10/2023 POSTERIOR PITUITARY HORMONES Classically, posterior pituitary hormones are not produced by neurohypophysis since they are released by nerve endings of hypothalamus; they are: Oxytocin: one of the most important hormones during pregnancy and labor. Vasopressin or ADH. The aminoacidic sequence of these two hormones is very similar; they are both composed by 9 amino acids and only 2 amino acids are different; they are protein hormones, and their receptors are transmembrane receptors, since only lipid-soluble hormones can cross the membrane, entering the cell and interacting with receptors in the cytoplasm or in the nucleus. electrolite balance can be affected by high presence of oxytocin Oxytocin Oxytocin is produced in the hypothalamus, in neurons located in the paraventricular nucleus; they are precursor peptides then activated and secreted by nerve endings that terminate in the posterior pituitary. There are other extra sites of production of oxytocin: Placenta Luteal cells of the ovary Endometrium However, the physiological meaning of these extra sites has not been really understood. Oxytocin interacts with specific receptors on the membrane of the cell, GPCR coupled with G-α-q subunit, leading to the final effect, the myometrium contraction, an important event during labor. Oxytocin binds its receptor, activating PLC that will cut the membrane phospholipids producing IP3 and DAG; then, IP3 will bind its receptor present at the level of sarcoplasmic reticulum mediating the release of intracellular Ca2+ that through the interaction with specific proteins will induce the activation of contractions in the myometrium. Oxytocin also increases local prostaglandins production, which further stimulates uterine contraction. There are oxytocin receptor antagonists useful to prevent pre-term birth. prostaglandins interact with receptors that are similar to those The sites of action of oxytocin can be: of oxytocin At peripheral level: Uterus → a peak of oxytocin occurs during the 3rd trimester of pregnancy; the physiological role is represented by the stimulation of the frequency and force of uterine contractions in order to promote the progression of the labor and to favor the dilatation of the cervix. Breast → oxytocin promotes the contraction of the myoepithelium that surrounds the alveolar channels in the mammary gland to allow the process of milk ejection. At central level: 15  Brain, CNS → oxytocin acts as regulator of trust, empathy (increases), anxiety and fear (reduces) and it is also important for the well-being of the CNS; in fact, the oxytocin released during the labor is involved in creating the bonding between the mother and the baby child, but also for communication. In autistic patients it has been reported a reduction in the oxytocin secretion and there are lots of studies in which scientists tried to inject oxytocin in order to promote the communication; the result was that oxytocin administered by inhalation could promote social interaction, reducing the social- communication deficit, one of the main features of anti-depressant effect, ansia autistic patients. However, this is not an optimal strategy since tolerance to the effects of oxytocin develops very rapidly, so it seems to have a pro-social activity during acute administration, but in the long-term treatment oxytocin loses its effect. Oxytocin can act as: Factor that reduces stress hormones (prepares the fetus to environmental shocks). Regulator of empathy. It reduces anxiety and the response to stressful stimuli. It promotes the bonding between the mother and child. Clinical indications 1. Induction of labor: it is administered when there is a risk for the mother to continue the pregnancy and generally it is used to favor the dilatation of the cervix in order to facilitate the progression of the labor. 2. Reduction of uterine hemorrhages, in order to favor the contraction of the myometrium. It is administered by an intravenous or intramuscular infusion, because of its nature of peptide; if it is administered orally, it would be degraded by proteases, so it wouldn’t be effective. It has a very short half-life (12-15 min). OXT adverse effects It is administered during labor inside the hospital because there is the possibility of potentially fatal adverse effects, such as the effect of (1) hyperstimulation of uterine contraction that would lead to the rupture of the uterus and to fetal distress; moreover there can be the possibility to have (2) hypertension (due to vasoconstriction) and also (3) water retention and toxicity because of its similar structure to vasopressin → the administration of high doses during labor could induce the activation not only of oxytocin receptor but also of vasopressin receptors (due to the similarity in structure). OXT analogues There are some analogues, characterized by a longer half-life; unfortunately, they are endowed with some important side effects because they remain longer in the blood circulation. 16 OXT receptor antagonist Atosiban is a synthetic peptide used to prevent pre-term labor; it blocks the receptor after the interaction, inhibiting the oxytocin signaling pathway; it can be used only during labor inside the hospital. It inhibits the release of IP3, reducing the release of calcium and so the contraction of myometrium. Its effect is exerted very rapidly, within 10 minutes and after the administration there will be a rapid relaxation of the muscle of the uterus → useful during emergencies of premature labor. Clinical indications → delay imminent pre-term labor in pregnant women with: Regular uterine contractions of at least 30 seconds duration at a rate of ≥ 4 per 30 minutes A cervical dilation of 1 to 3 cm (0-3 for nulliparas) and effacement of ≥ 50% Age ≥ 18years A gestational age from 24 until 33 completed weeks A normal fetal heart rate Adverse effects: Gastrointestinal effects: nausea Headache Dizziness Tachycardia Urinary tract infection Another important aspect is that Atosiban is useful during IVF; in particular, during embryo transfer (that can be problematic for the success, because the uterus starts to have contraction after the effective embryo transfer, since it is stimulated by the procedure), the injection of Atosiban reduces the contractility of myometrium and it could improve the success in the step of embryo transfer during IVF → in this way, there will be an optimal intrauterine environment when the embryo is transferred. There are lots of studies that suggest this technique. 17  THYROID HORMONES Thyroid gland is in the anterior neck; it is composed by follicles made of a layer of follicular cells and filled with colloid, a substance made of proteins and thyroid hormones; there are some other cells, parafollicular cells responsible for the production of calcitonin, important for calcium metabolism. Follicle = follicular cells with colloid + parafollicular cells. In the follicular cells it is possible to distinguish a basolateral side and an apical side; they uptake iodide and amino acids from the blood on the basolateral side and inside the cells amino acids are necessary to produce thyroglobulin and then thyroperoxidase enzymes. Thyroglobulin is a protein able to transport thyroid hormones and to produce thyroperoxidase enzymes. The follicular cells subsequently take up iodinated thyroglobulin from the follicles by endocytosis, extract thyroid hormones from it with the help of proteases and subsequently release thyroid hormones into the blood. There are 2 hormones: T3 → triiodothyronine: it has 3 iodine atoms, made of 1 molecule of Iodothyronine and 1 of Diiodotyrosine T4 → thyroxine: it has 4 iodine atoms, made of 2 molecules of Diiodotyrosine. And other derivatives: Diiodotyrosine (DIT): 2 iodine atoms Monoiodotyrosine or Iodothyronine or (MIT): 1 iodine atom Mechanism of production 1. Iodination of thyroglobulin: Iodine ingested in the diet reaches the circulation in the form of iodide ion (I−); Iodide is taken up by the follicular cells from the blood and it is transported into the colloid thanks to pendrin, a transporter of iodide; iodide will be the substrate of tyroperoxidase enzyme which, together with thyroglobulin, induces the organification of iodine, producing MIT and DIT, just prior to its extracellular storage in the lumen of the thyroid follicle. 2. The iodinated amino acids are converted into T3 and T4 through a coupling of 1 MIT and 2 DIT to produce T3 and 2 DIT to produce T4. 18  3. T3 and T4 are then taken up by the follicular cells through endocytosis and then through the fusion of the endosome + the lysosome, thanks to proteolytic enzymes, the hormones will be released into the blood. The active form of the hormone is T3 and the conversion of T4 into T3 is mediated by deiodinases, which exist into 3 different isoforms: D1, D2, D3, characterized by tissue-specificity. In some pathological conditions → overexpression or downregulation of these enzymes; Hyperthyroidism→ upregulation of D1, so there is a massive conversion of T4 into T3. Hypothyroidism → downregulation of D1, so there is a lower activity of T3. The receptors of thyroid hormones are nuclear receptors, even if they are peptides, since thyroid hormones have a transmembrane transporter that allows the entry of T3 inside the cell. T3 receptor is located in the nucleus, and it does not exist as a monomer but only as a heterodimer; indeed thyroid hormone receptor is coupled with another receptor: retinoid acid receptor, (RXR) able to interact with specific sequence of DNA, after the binding with thyroid hormones. It is possible to distinguish the 4 domains: AF1 and AF2: domains regulated by co-activators and co-repressors that stimulate or not the transcription of specific genes. DNA-binding domain (DBD), important to bind sequence of the DNA named as HRE, hormone responsive element ligand-binding domain (LBD). Thyroid gland is under the control of the hypothalamus. In particular, the hypothalamus releases TRH, which is considered the stimulus for inducing an increase in the release of TSH from the pituitary gland, which then will stimulate the thyroid gland to produce Thyroid hormones, T3 and T4. To summarize: Hypothalamus → TRH → pituitary gland → TSH → thyroid gland → T3 T4. There is an inhibitory feedback control of this axis; when thyroid hormones levels are high in the plasma, the release of TRH from hypothalamus is inhibited; on the opposite, when the levels of thyroid hormones in the plasma are low, hypothalamus is activated and it releases TRH, stimulating the release of TSH. The effects of thyroid hormones are quite complex; they exert their function in all the cells of the body, and they are involved in different types of activities: Control the rate of cellular metabolism. Thermogenesis. Influences the functioning nearly every cell in the body. 19  They affect the heart, the liver, kidneys, and the skeleton muscles. Cardiovascular effects: T3 increases cardiac output, cardiac ejection, it increases the force of contraction of the heart; it also reduces vascular resistance. They can have crucial effects during the neurogenesis, which is the development replacementn of CNS and in fact, the lack of thyroid hormones is the cause of a disease called Cretinism; for this reason, when the baby is born, it is tested for the proper production of thyroid hormones and if there is a dysfunction of the thyroid gland, the baby undergoes a replacement therapy, because these hormones are fundamental for the correct development of the nervous system. Iodine is an essential nutrient crucial during the neurodevelopment for the migration of GABAergic and glutamatergic neurons; if this process does not occur → important cognition deficit, like cretinism. T3 stimulates beta-adrenergic transmission. There are 2 pathological conditions associated with thyroid hormones: 1. Hypothyroidism: associated with low levels of thyroid hormones 2. Hyperthyroidism: associated with high levels of thyroid hormones Hypothyroidism There are some important symptoms: Bradycardia: thyroid hormones are important for the stimulation of beta-adrenergic transmission. Sense of asthenia, fatigue. Weight gain: when thyroid hormones are low, there is an impaired metabolism. Irregular menses and infertility: this condition is common also in hyperthyroidism. One example of hypothyroidism is Hashimoto’s disease, an autoimmune disease direct to cells of thyroid gland, so thyroid gland is progressively destroyed by the autoantibodies. In this condition there are low levels of T3 and T4 in the blood but high levels of TSH, because the mechanism of feedback inhibition does not work. Therapy In this case it is possible to undergo a replacement therapy, by injecting analogues of thyroid hormones like Levothyroxine and Thyroxin (gold standard) Hypothyroidism is a common condition during pregnancy, because there is the possibility to have an increase of TBG, so the strategy is to increase the dosage of thyroxin. Adverse effects are: Atrial fibrillation Risk of osteoporosis 20 Hyperthyroidism This condition is characterized by: Heat, the skin is moist because of the increased metabolism. Loss of weight. Increased appetite. Tachycardia, because of the hyperstimulation of the beta-adrenergic transmission. Nervous System effects: insomnia, difficulty to remain still, anxiety. Muscles can be tremulous. Increased frequency of bowel movements. Angina, arrhythmias, and heart failure. In this condition the levels of TSH are zero, because there are high levels of thyroid hormones. In Graves’s disease there are antibodies that bind and activate TSH receptor; this ectopic continuous activation of TSH receptor produces an increase in the production of thyroid hormones. Therapy In this case it is possible to use anti-thyroid drugs to reduce the excess of thyroid hormones, so all the steps involved in the production of these hormones (iodide uptake by the cells, organification, coupling and all the mechanisms). It can be possible to use compounds that reduce the hyperstimulation of beta- adrenergic transmission, like beta-blockers. Iodide is also used since it impairs the entry of iodine inside the cell. There are 3 main drugs: Antithyroid drugs that interfere with the synthesis of thyroid hormones. Ionic inhibitors, that block the iodine transport mechanism. High concentrations of iodine, that decrease the release of thyroid hormones from the thyroid gland. Radioactive iodine, which destroys the cells of thyroid gland with ionizing radiation. Antithyroid drugs 1. Propylthiouracil (PTU): half-life of 75 minutes; PTU interferes with the incorporation of iodine, blocks the conversion of T4 into T3 in the periphery finally inhibiting the coupling and deiodination of T4 into T3; it is more potent than MMI. Not recommended in children because of the risk of severe hepatotoxicity and death; it is used only at high doses used in the treatment of thyroid storm in which there are high levels of thyroid hormones that increase a lot the frequency of the heart leading to potential lethal effects. PTU is used when a patient develops side-effects to carbimazole. 2. Methimazole (MMI): half-life of 4-6 hours; it does not have the same effects as PTU. It is very useful, for example in cases of conditions that have to be rapidly treated. 3. Carbimazole: half-life of 4-6 hours; it is the drug of choice. 21 PTU mechanism of action PTU: interfering with the incorporation of iodine inhibits the coupling of these iodotyrosyl residues to form iodothyronines and inhibits the peroxidase enzyme. Adverse effects: The incidence of side effects from propylthiouracil and methimazole as currently used is relatively low. Agranulocytosis is the most severe reaction. Cutaneous side effects such as urticarial skin rash (the most common). Thyroid hormones and fertility Thyroid hormones are useful to maintain the normal reproductive system and so Thyroid hormones dysfunction such as the conditions of hypo/hyperthyroidism could lead to impairments in fertility. Prolactin is under the control of dopaminergic neurons that exert an inhibitory activity, but they are under the control of TRH produced by the hypothalamus. During the condition of hypothyroidism, there is the lack of inhibitory control of Hypothalamus-Pituitary Axis and so the hypothalamus is stimulated to produce TRH that will then stimulate the production of TSH that will stimulate thyroid gland activity; one of the effects could be the onset of hyperprolactinemia and some impairments in the release of GnRH that should occur in a pulsatile way; these two effects lead to a delay in LH response → inadequate corpus luteum, leading to impairments in the menstrual cycle and irregular menses, common in people affected by hypothyroidism. Starting from the fact that the inhibitory feedback exerted by thyroid hormones on the hypothalamus lacks, the hypothalamus is ready to produce TRH that acts on the pituitary for the production of TSH that will affect the thyroid for the production of thyroid hormones. There is a high similarity in terms of structure between TSH and Gonadotropins, FSH and LH; so, when there is a condition characterized by increased levels of TRH, that stimulates TSH, leading to some impairments in the production of FSH and LH. This can happen in males → hypothyroidism in males induces an increase in hyperprolactinemia and so there can be an alteration in the production of LH. Next, there can also be impairments produced by FSH; high levels of TSH, due to similarity to FSH, can induce the production of sperm and the maturation of testis. There are also metabolic problems like what happens in adipose tissue: it can increase estrogen production. 22 Function of thyroid hormones in testes T3 regulates the maturation and growth of testis, controlling both the activation of Leydig and Sertoli cells; hypothyroidism generally induces a delay in sexual maturation and so there can be lots of problems related to hormonal dysfunction. There is a strong link between thyroid hormones and vitality of spermatozoa; when there are impairments in the levels of thyroid hormones, there could be problems in the vitality of spermatozoa. Lecture 6 - 19/10/2023 ESTROGENS Estrogens are the primary female sex hormones, responsible of the development and the regulation of the female reproductive system and secondary sex characteristics during puberty. The three major endogenous classes are estrone (E1), estradiol (E2), most prevalent of these, and estriol (E3). Structurally, they are formed by a phenolic A ring, with a selective high affinity to both receptors. Sometimes, substitutions on ring C and D may be tolerated, like the most important ethinyl substitution on C17 position, that greatly increases the oral potency, by inhibiting the first-pass hepatic metabolism. Alkil substitutions on A ring can impair the binding. The oral administration of estrogens is an alternative method to transcutaneous patches, that exploit their lipidic nature, but are not as practical as the oral route. Steroidal estrogens arise from androstenedione or testosterone by aromatization of A ring, in fact blocking the aromatase can reduce the amount of estrogens in the body. The aromatase enzyme uses NADPH and molecular oxygen co-substrates, but in this reaction is also essential the activity of flavoprotein NADPH-cytochrome p450 reductase; both proteins are localized in the endoplasmic reticulum. 23 Biosynthesis Ovaries are the principal source of estrogens in pre-menopausal women, in particular theca cells and granulosa cells. LH acts on theca cells through receptors that are coupled to the Gs- adenylyl-cyclase-cyclic AMP pathway to increase cholesterol transport into the mitochondria, where androgens precursors are produced. FSH stimulates CYP19 production in granulosa cells, that are responsible for androgens conversion to estrogens. In post-menopausal women adipose tissue stroma is the principal source of estrogens, starting from dehydroepiandrosterone secreted by adrenals, which is converted to estrone. Neuroendocrine control of menstrual cycle The hypothalamus is the starting point, where the neural clock in the arcuate nucleus works as a pulse generator composed of neurons, that at regular time intervals mediates the pulsatile secretion of GnRH. This kind of secretion is synchronized to the LH peak. Then, GnRH stimulates the anterior pituitary to release gonadotropins, which will act at multiple levels, for example in the ovaries they stimulate estrogens production. During menstrual cycle estrogens rise in the follicular phase and in ovulation, together with the peak of LH. Effects of estrogens and progesterone Estrogens and progesterone production by ovaries regulate corresponding events in the fallopian tubes, uterus, cervix, and vagina. Physiologically, the increase of progesterone leads to many changes in the uterus to allow 24 implantation, which is essential to establish a pregnancy; however, when pregnancy doesn’t occur, the endometrium degenerates and sheds causing the menstrual cycle. In fallopian tubes the proliferation and differentiation are inhibited by progesterone, while muscular contractility is increased by estrogens but is decreased by progesterone, affecting the transit of the oocyte to the uterus. Estrogens increase the amount of cervical mucus facilitating the sperm penetration of the cervix while the progesterone does the opposite effect, by increasing its viscosity. As far as the uterus, the rhythmic contraction of uterine myometrium is favored by estrogens, while the progesterone diminishes this activity. Estrogens have a positive effect on bone mass, increasing calcium absorption, alter the number of metabolic pathways that affect the clotting cascade, increasing coagulation factors II, VII, IX, X and XII and decreasing anticoagulation factors protein C, S and antithrombin III. Estrogens receptors Estrogens alpha and beta receptors are in cytoplasm, acting as transcription factors. They have a great homology and are differently distributed within tissues, because the alpha ones are most abundantly expressed in ovaries, uterus, and vagina, while the beta ones are mainly in lungs, brain, bones, and vasculature. For what concerns the structure, they are composed of classical domains, like the ligand-binding domain, the DNA-binding domain, and the specific regions AF1 and AF2, which can recruit co-activators and co-repressors. Once the hormones are bound to the receptor, the complex dimerizes and penetrates the nucleus where it can interact with specific DNA sequences called ERE, affecting the transcriptional machinery. The classical way includes the complex receptor- ligand that leads to the transcriptional activity, but there are other mechanisms like the ligand-dependent and the ligand-independent pathways, which mediate rapid effect of estrogens, that doesn’t require the translation of proteins but the activation of kinases that perform phosphorylation. Estrogens can be administered in their natural form, so estradiol, estriol and estrone, but also in a synthetic form. In this case they are differentiated between steroidal, like ethinylestradiol, found in contraceptive pills, obtained by adding ethinyl to the C ring in position 17, and mestranol, and non-steroidal, like diethylstilbestrol, hexestrol, dienestrol. 25 For what concerns pharmacokinetic properties, estrogens have a lipophilic nature, and are available for oral, parenteral, transdermal, and topical administration. Oral administration has a low bioavailability, so it’s better to conjugate estrogens with ethinyl estradiol, while administering estradiol through transdermal patches provides a slow sustained release of the hormone and a systemic distribution, with more constant blood levels than oral dosing. Estradiol is also available as a topic emulsion. So, the use of estrogens is implied in hormonal contraception (contraceptive pill) and hormone replacement therapy, during menopause, where it’s better to use transdermal patches with no natural hormones. Moreover, it’s possible to distinguish between two types of molecules: selective estrogen receptor modulators (SERM), like tamoxifen, raloxifene and toremifene, and selective estrogen receptor down-regulators (SERD) or antiestrogens, like clomiphene, fulvestrant or inhibitors of aromatase. SERMs Selective estrogen receptor modulators have a tissue selective action because they both perform a beneficial estrogenic activity in some tissues, like bones or liver, and an antiestrogenic action in some others, preventing harmful effects of estrogen in breast, for example in case of hormone-dependent cancer, where tumor cells overexpress on their surface estrogen receptors, so in that way the abnormal proliferation of cancer cells is reduced by blocking receptors. Tamoxifen, used as an adjuvant during chemotherapy in the treatment of breast cancer, has increased the survival rate of the women affected. This drug maintains the effects of estrogen in bones, lowering fracture risk, but also in liver, reducing the amount of LDL and cholesterol; however, in blood it can induce deep venous thrombosis. At uterine level, it increases the endometrial proliferation, but in breast it exerts an antiestrogenic effect, acting as an antagonist, while it performs an agonist activity in bones, liver, blood, and uterus. Tamoxifen’s adverse effects are the risk of endometrial cancer, contrary from raloxifene, deep vein thrombosis and pulmonary embolism, nausea, vomiting, hot flashes. It is generally used to treat but also prevent breast cancer in pre- and post-menopausal women. The ability of SERMs to induce at the same time agonistic and antagonistic effects in different tissues depends on the ratio between co-activators and repressors typical of a particular tissue. Normally, estradiol recruits co-activators and the histone acetyl transferase, inducing the relaxation of chromatin and the activation of transcription, while antagonists, like Tamoxifen (T), bind the receptor and stimulate a change in conformation with the recruitment of co-repressors and the histone deacetylase, that leads to a compaction of chromatin, inhibiting the transcription. 26 SERDs (antiestrogens) They have a selective action, performing both agonist and antagonist functions, but they also downregulate estrogen receptors, performing a total antiestrogenic activity by inducing the reduction of receptors expression, so that a cell is not responsive anymore to them. Fulvestrant binds the ER at the level of the unfolded H12 helix, inhibiting co-activators recruitment. Then the dimerization is impaired, so that the complex can’t bind ERE on DNA, and the receptor’s degradation is accelerated. Fulvestrant is generally used to treat breast cancer in post-menopausal women. Clomiphene inhibits estrogen receptors in hypothalamus, increasing GnRH secretion, and so FSH and LH production, resulting in ovulation. This happens because the hypothalamus doesn’t respond to the negative feedback exerted by estrogen, even if their blood level is high, so it is stimulated to produce GnRH. For this reason, clomiphene is always administered in assisted reproduction, to favor ovulation in females affected by severe anovulation. The adverse effects comprehend ovarian hyperstimulation syndrome, polycystic ovary, multiple pregnancies, hot flashes, vomiting and nausea. Aromatase inhibitors (AIs) selectively block estrogen production. They can be steroidal, type I agents substrate analogue, that irreversibly inactivate aromatase (formestane and exemestane), or non-steroidal, type II agents, that interact irreversibly with the heme groups of CYPs (anastrazole, letrozole and vorozole). These antiestrogens inhibit the conversion of androstenedione and testosterone to estradiol, reducing estrogen content. Among adverse effects there are hot flashes, vaginal bleeding, endometrial cancer, venous embolic events, loss of bone mineral density, osteoporosis. They’re generally used to treat breast cancer in post- menopausal women, while pre-menopausal women with the same condition are treated with tamoxifen. This difference is explained by the fact that in post- menopausal women the primary source of estrogen are non-ovarian tissues, like fat, liver, muscle, brain, breast, because ovaries don’t work, and HPA axis, differently from pre-menopausal women, where the HPA axis works very well. So, by using AIs, the hypothalamus senses a decrease of estrogen, while estrogen levels in blood are still high, so it produces GnRH that causes the release of gonadotropins, which act on ovaries that produce estrogen. So, it’s very difficult to reduce estrogen levels in pre- menopausal women by using AIs, contrary from what happens when it is used in association to GnRH antagonist, to suppress both hypothalamic and pituitary activities. Treating breast cancer includes SERMs like Tamoxifen, Toremifene and Raloxifene, and SERDs like Fulvestrant. In pre-menopausal women the 27 medical treatment can be coupled with surgical oophorectomy, while in post-menopausal women, it’s better to use aromatase inhibitors, like anastrozole and letrozole. PROGESTERONE AND PROGESTINS Progestins have a biological activity like progesterone. They include progesterone, 17 alfa-acetoxyprogesterone derivatives in the pregnane series, 19-nortestosterone derivatives in the estrane series, and norgestel with its related compounds in the gonane series. These compounds have a highly dependent activity on specific substituent groups, like the nature of C17 in D ring, the presence of a C19 methyl group and the presence of an ethyl group in position C13. Progestogens can be classified in: As far as biological activity, these compounds are classified according to progestational activity, androgenic activity, estrogenic activity and antiestrogenic activity. Comparing them to estrogen, they seem to have some opposite effects, but they also have similarities to androgens, acting sometimes in the same way, like levonorgestrel. The progesterone is secreted by ovarian corpus luteum during the second half of the menstrual cycle, by stimulation of LH, that acts via its GPCR. After fertilization, the trophoblast secretes hCG, which stimulates LH receptors to sustain corpus luteum in progesterone production. Then, both estrogen and progesterone are secreted by the placenta until birth. The physiological actions of progesterone are: Neuroendocrine control of GnRH pulses decreases the frequency of GnRH pulses to suppress gonadotropins release and reset the hypothalamic-pituitary-gonadal axis, making the transition from luteal phase to the follicular one. Conversion of the endometrium to secretory stage for implantation, with the reduction of endometrial proliferation promoted by estrogens, leading to the development of secretory glands. When the fertilization doesn’t occur the levels of progesterone decline, causing the end of the cycle with the onset of menses. Affection of vaginal epithelium, cervical mucus, mammary glands, where progesterone brings out a proliferation of the acini of the mammary gland, and CNS, acting as a depressant and hypnotic, helping 28  patients to sleep when administered before bedtime. Also, as far as metabolism, it increases basal insulin levels. Pharmacology of progesterone and progestins The receptor has two isoforms, alpha and beta, with similar domains to recruit co- activators and co-repressors. Also, there’s another way in addition to the activation of transcriptional machinery, to obtain rapid effects of progesterone. The progesterone receptor is present primarily in the nucleus in an inactive monomeric state bound to HSP90, HSP70, and p59. When receptors are phosphorylated and subsequently form dimers that bind with high selectivity to PREs located on target genes. The progesterone undergoes rapid first- pass metabolism when administered through oral route. It’s also available in oil solution for injection, as a vaginal gel, as a slow-release IUD for contraception, and as a vagina insert for ART. When it’s used as a contraceptive or during hormone replacement therapy, it can help in uttering bleeding, endometriosis, reducing endometrial proliferation, premenstrual syndrome tension, ART luteal support, to prevent recurrent miscarriages and preterm labor, by reducing myometrial contractility. Luteal support is mandatory in IVF cycles, because many protocols are based on GnRH agonists or antagonists, to avoid the precocious rupture of follicles when they have not achieved maturity yet. The protocol can induce progesterone deficiency due to the reduction of LH, but it has also improved pregnancy outcomes, even though some progestins, like dydrogesterone, should not be administered because they’re at risk of causing congenital heart defects. Lecture 7 - 25/10/2023 Progesterone exerts some effects on the reproductive system: for example, on the mammary gland (to induce enlargement of breasts), but also a physiological action from metabolic point of view and it also has an important role in the second part of the menstrual cycle, because progesterone is produced by corpus luteum and prepares the uterus to receive the implanted egg. It has some effects that are opposite to those induced by estrogens. Clinical indications Progesterone, and in general progestins, are used as contraceptives, in particular there are 2 different approaches: combined contraceptive pills (both estrogen and progesterone) and the progesterone only pills. It is used also in menopause (hormonal) replacement therapy, in dysfunctional uterine bleeding, in endometriosis, in premenstrual syndrome, for preterm labor prevention, and in miscarriage and in ART→ in this last one, the role is to induce the luteal support. 29  Role of progesterone in ART Progesterone is used to induce luteal support. In fact, during IVF protocols, it’s possible to use GnRH agonist or antagonist, and this therapy induces the suppression of LH surge → the role of these compounds is to prevent a precocious rupture of the follicle. So, the treatment with GnRH agonist and antagonist can lead to lack in the production of LH. In some ways, the administration of progestins can improve pregnancy outcomes, since it induces the relaxation of the uterus, which is important to prevent higher contractility of the myometrium and so on. Role of progesterone in recurrent miscarriage Progestins can also be helpful during recurrent miscarriage: it enhances implantation, reducing effect of estrogens in the myometrium and, in particular, it reduces the myometrial contractility and cervical dilatation, that are effects important to maintain the closure of the cervix. Role of progesterone in preterm labor prevention There are different mechanisms of action and finely tuned specific pathways activated by progesterone. By interacting with its receptor, the progesterone: o Inhibits the degradation of type 1 collagen in the cervix, so it reduces the softening of the cervix itself, the dilation of the cervix. o Responsible for the control of the release of prostaglandins, so autocrine and paracrine control. o Inhibits the expression of contraction-associated genes in myometrium, so inhibits the contractility; this last aspect is very important to continue the pregnancy. Progesterone helps to prolong the pregnancy up to term or late preterm and decreases incidence of neonatal morbidity of neonate. Antiprogestins and progesterone receptors modulators Progestins used for contraception and also to reduce preterm labor, taking advantages from its effect on the contractility and dilatation of the cervix. However, there are compounds defined as antiprogestins and progesterone receptor modulators: these are mifepristone (1), also called RU 38486 but mainly RU-486, and ulipristal acetate (2). 1. Mifepristone is a derivative of the progestins, and it’s considered as progesterone receptor modulator, since it exerts some antagonistic effects by interacting with progesterone receptor and recruiting corepressors. It is a competitive receptor antagonist for both PRs. Progesterone is a lipid soluble compound, so it has a soluble receptor in the cytoplasm, and the mechanism of action involves the activity of transcription factors. This receptor, after the binding, can recruit co repressors and give an antagonistic effect (while when co activators are recruited, there is an agonistic effect). Mifepristone acts as antagonist because preferentially tends to recruit co repressors, although this doesn’t happen in all tissues. The effects of mifepristone depend on the schedule of administration: When administered during the early stages of pregnancy → it blocks progesterone receptors in uterus and there can be the detachment of the blastocyst, which decreases hCG production; it also decreases in progesterone secretion from corpus luteum, which accentuated decidual breakdown. It also causes cervical softening, which facilitates the expulsion of the blastocyst. The final effect is abortion. When administered for one or several days in the mid- to late-luteal phase → it impairs development of secretory endometrium and produces menses. It can be used as a contraceptive emergency. 30  It has a good availability, an oral administration. Plasma t1/2 of 20/40h, it presents a hepatic metabolism, and it is eliminated in the feces. The clinical indications are: First, termination of pregnancy → mifepristone is administered with misoprostol or another prostaglandin for termination of early pregnancy. It is also used to produce medical abortion, in fact, prostaglandins are used to increase the contractility of the myometrium to facilitate the expulsion of the blastocyst. Misoprostol is usually used for labor induction. Indeed, it causes uterine contraction and ripening of the cervix. To have abortion: mifepristone, then after 48h the prostaglandin (to eliminate the blastocyst). The adverse effects are uterine clumps, nausea, vomiting, diarrhea, vaginal bleeding, and abdominal pain. 2. Ulipristal is a derivative of 19-norprogesterone, functions as a selective progesterone receptor modulator, acting as a partial agonist at PRs. It inhibits ovulation and prevents fertilized egg from attaching uterus, and it is essentially used as emergency contraception. Mifepristone has a quart long half-life (around 20h), while Ulipristal remains effective up to 120h → it can be useful to be active also after 5 days of the potentially dangerous intercourse, so it’s more versatile and allows a higher protection. Uses of estrogens and progestins Hormonal contraception → Both estrogens and progestins are used. We have 3 types: combination oral contraceptives (COCs), progestin only contraceptives (POCs) AND intrauterine devices. 1. COCs: combination of both estrogens and progestin. Almost all of them contain ethinyl estradiol as main component and derivative of nortestosterone as progestin. They are characterized by a very high effectiveness, 99.9%. For what regards the content of the preparation they contain from 20 to 50 μg of product, most of them 30/35 μg. Those pills that contain lower doses, so