Instructor: Patrick M. Woster, Ph.D.
Biosynthesis of Sex Hormones
The biosynthesis of the steroid sex hormones begins with cholesterol, as shown in the figure below. Side chain cleavage of cholesterol produces the intermediate pregnenolone, which is the immediate precurson of progesterone. Through a series of steps, pregnenolone is converted to the androgen precursor androstenedione, and then to testosterone. Androstenedione and testosterone are in turn converted into the estrogens estrone and estradiol, respectively. This reaction is catalyzed by the enzyme aromatase. Estradiol is further converted to estriol by the enzyme 16-α-hydroxylase. The pathway shown below is meant to be a summary, and the actual biosynthesis of these hormones is quite complex.
Estrogens
As was mentioned above, the three major estrogens in humans are estrone, estradiol and estriol. Estradiol is the most potent endogenous estrogen, and has the highest affinity for the estrogen receptor. On oral administration, it undergoes conjugation in the intestine and oxidation in the liver, and thus has low oral bioavailability. It is also highly protein bound, and stimulates the synthesis of sex hormone binding globulin (SHBG), to which it binds very well. Estradiol is also rapidly converted to the less effective estrogen estrone by the enzyme estradiol dehydrogenase. One strategy to increase the duration of action of estrogens is to add a substituent at the 17-position, as in ethinyl estradiol and mestranol. Another strategy is to form esters, as in estradiol-17-ß-valerate and estradiol-17-ß-cyclopentanepropionate. The ethinyl derivatives are generally used orally, while the ester formulations are often administered by intramuscular injection. These preparations may be considered as pro-drugs, since they must be hydrolyzed for activity, and they have exyended duration of action.
The conjugated estrogens are a mixture of water-soluble estrogen sulfate esters that were originally isolated from pregnant mare's urine (hence the trade name Premarin®). There are a total of 10 known conjugated estrogen compounds, and 9 of these can now be produced from plant sources (soy and yams).
There are two estrogen receptor (ER) subtypes, known as α and β, and they are encoded by two separate genes (ESR1 on chromosome 6 and ESR2 on chromosome 14 respectively). Hormone binding to the receptor triggers migration of the receptor from the cytosol into the nucleus, dimerization of the receptor, and binding of the receptor dimer to specific sequences of DNA known as hormone response elements. The DNA/receptor complex then recruits other proteins which are responsible for the transcription of downstream DNA into mRNA and finally protein. Hormone activated ERs form homodimers (ERαα or ββ) or heterodimers (ERαβ). Different ligands may differ in their affinity for α and ß isoforms of the estrogen receptor. Thus, 17-ß-estradiol binds equally well to both receptors, estrone binds preferentially to the α receptor, and estriol prefers the ß receptor. Subtype selective estrogen receptor modulators (SERMs) preferentially bind to either the α- or β-subtype of the receptor. Additionally, the different estrogen receptor combinations may respond differently to various ligands which may translate into tissue selective agonistic and antagonistic effects. The same ligand may be an agonist in some tissues, and an antagonist in other tissues. Tamoxifen is an antagonist in breast and is therefore used as a breast cancer treatment, but acts as an ER agonist in bone and an agonist in the endometrium. In the absence of hormone, estrogen receptors are largely located in the cytosol.
There are a number of non-steroidal compounds that have potent estrogenic activity. Many of these are derivatives of trans-stilbene, the most important of which was diethylstilbestrol (DES). The trans isomer of DES is about 10 times as potent as the cis, because it is more similar to the structure of estradiol. DES is no longer used due to a higher incidence of uterine cancer in patients taking the drug. A second class of non-steroidal estrogen is the xenoestrogen or environmental estrogen class. These compounds occur in the environment, either naturally or as a by-product from plastics, pesticides, etc. Environmental estrogens are also termed estrogen disruptors, since they have activity at estrogen receptors in humans. Many environmental estrogens are from plant sources (the phytoestrogens). Genistein is a natural isoflavone that is found in soy products, and some studies have indicated that a diet rich in soy products can reduce the incidence of breast cancer through antioxidant activity. By contrast, bisphenol A is released from plastic bottles and can liners when they are scratched or heated, and has significant estrogenic activity. Some researchers believe that reproductive system changes in animals are due to exposure to environmental estrogens, and these may also cause early puberty.
Agents that can antagonize the effects of estrogen at the receptor are of interest because they can modify reproductive processes, or because they are useful in the treatment of estrogen receptor-dependent breast cancer. The triphenylethylenes bind tightly and persistently to the estrogen receptor, but the resulting complexes either do not translocate into the nucleus, or do not bind properly to the estrogen response element. Clomiphene is a partial estrogen agonist, and is used in the treatment of infertility. It causes the release of hormones such as follicle-stimulating hormone and luteinizing hormone, causing ovulation. The use of clomiphene has been associated with multiple births. As was mentioned above, tamoxifen has antiestrogenic activity in breast tissue, and is thus used in the treatment of breast cancer. It also has weak agonist activity in bone, and can also be used to treat osteoporosis.
Recent advances in the understanding of estrogen receptor molecular pharmacology have facilitated the development of selective estrogen receptor modulators (SERMs). These compounds nafoxidine and raloxifene are rigid analogues of the triphenylethylene class. Raloxifene, for example, produces antiestrogenic activity in breast tissue, but not in the uterus.
Aromatase inhibitors have utility in the treatment of estrogen-dependent breast cancer, and in the control of reproductive functions, because they prevent the conversion of androstenedione and testosterone to estrone and estradiol, respectively. A number of steroid derivatives typified by 10-ß-propynylandrostenedione and 7-α-aminophenylthioansrostenedione act as "suicide substrates", and irreversibly inhibit aromatase. Two triazole aromatase inhibitors, anastrozole and letrozole, are effective aromatase inhibitors that are used clinically to treat advanced breast cancer in postmenopausal women who have failed tamoxifen therapy.
Progestins
Progesterone has long been known to suppress ovulation during pregnancy, and thus has natural contraceptive activity. However, progesterone itself has relatively low bioavailability,and was primarily used by parenteral injection, prompting medicinal chemists to search for a suitable synthetic progesterone analogue. The first synthetic progesterone derivative used for female contraception, norethidrone, was synthesized by Carl Djerassi in 1961. Djerassi, who was a professor of chemistry at Wayne State University from 1952 to 1959, is commonly known as "the father of the pill". The term "the pill" was coined by Aldous Huxley in his book Brave New World.
Addition of a 17-α-acetoxy group to progesterone produces an increase in activity, and is effect was enhanced when a substituent was added at the 6-position. The first of these so-called pregnanes, the analogue medroxyprogesterone acetate, is a comonly used progestin (Provera®, Depo-Provera®). Although medroxyprogesterone acetate is 86% protein bound in plasma, it has no affinity for SHBG. Addition of a 6,7-double bond enhances activity even further, as in megastrol acetate.
A second class of synthetic progestins, the 19-norandrostanes, also arose from the search for an orally active, bioavailable progestin. Medicinal chemists determined that the 19-methyl group was not necessary for progesterone-like activity, and the 17 acetyl group could be replaced by a hydroxyl, as long as a second substituent was present. The two most useful agents in this class, norethindrone and norethynodrel, are commonly used as the progestin component of birth control pills. Despite much research, only a handful of progestin antagonists have been discovered, the most famous of which, mifeprostone or RU486, interferes with the early stages of pregnancy.
Androgens
The earliest report of the isolation of a naturally occurring androgen occurred in 1931, when Butenandt and co-workers isolated 15 mg of androsterone from 15,000 liters of male urine. In humans, testosterone can undergo reductive metabolism through the enzyme 5-α-reductase, to form 5-α-dihydrotestosterone (DHT), or it can be oxidized to form androstenedione. As we have seen, it can also be converted to estradiol by the action of aromatase.
The physiological effects of testosterone and its metabolites 5-α-dihydrotestosterone (DHT) and estradiol (produced in small quantities in males) are mainly exerted in the male reproductive system (prostate and seminal vesicles), as well as muscle, bone and the brain. Testosterone preferentially binds to androgen receptors in muscle, bone and brain, while DHT prefers the androgen receptors in the genitalia, prostate, skin and hair follicles. Male pattern baldness is a phenomenon that is thought to be caused by DHT-mediated destruction of hair follicles. The effects of the androgens are mediated through the androgen receptor. The androgen receptor is maintained in an inactive complex with heat-shock protein 70 (HSP70) and HSP90, as well as other co-repressors. When a molecule of testosterone or DHT binds, the repressor proteins are released, the receptor dimerizes, and the complex translocates to the nucleus, where it binds to the androgen response element. At that time, co-activators and transcription factors bind to the complex, initiating transcription. One of the products of androgen-initiated transcription is prostate-specific antigen, which is commonly used to monitor or detect prostate carcinoma.
Testosterone was the first androgen used clinically, but it was expensive to produce (drug companies could isolate only 270 mg of testosterone from one ton of bull testes). In addition, testosterone is ineffective orally due to extensive first pass metabolism. These facts spurred efforts to discover a potent, orally active androgen that could be used to treat hypogonadism in males, and to produce an anabolic effect on skeletal muscle. It was soon discovered that addition of a 17-α-methyl group increased potency and imparted oral activity (as in the examples below), and that introduction of a 9-α-fluorine enhanced activity even further. As in the case of the estrogens, formation of testosterone esters increases the duration of action, since the ester must be hydrolyzed to form the active constituent.
Synthetic analogues of testosterone have a place in pharmacotherapy for thr treatment of male hypogonadism and for their anabolic effects. However, androgen replacement therapy is controversial, since it is possible it is associated with the promotion of androgen-dependent prostate tumors. It is virtually impossible to separate the androgenic and anabolic effects of synthetic steroids, which all bear a structural resemblance to testosterone. Three commonly used synthetic anabolic steroids, oxymetholone, oxandrolone and stanozol are shown below, but there are a number of other examples. The so-called "designer steroids" are often in the news because of their rampant abuse by professional atheletes. For this reason, agents such as desoxymethyltestosterone (DHT), tetrahydrogestrinone and trenbolone are on the banned substances list of the International Olympic Committee.
Inhibitors of 5-α-reductase have found utility in the treatment of benign prostatic hypertrophy, and in they join α-adrenergic antagonists such as tamsulosin (Flomax®) as a second treatment option.The compounds finasteride (Proscar®) and dutasteride (Avodart®) both act as mechanism-based inhibitors of 5-α-reductase. Finasteride is selective for type 2 5-α-reductase, and allows some DHT to form from type 1 5-α-reductase. Dutasteride inactivates both type 1 and type 2 5-α-reductase, producing a more complete inhibition of DHT biosynthesis. Both compounds are extensively metabolized by cytochrome P450, and as such the dose should be adjusted when a p450 inhibitor is co-administered.
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