Discovery and development of antiandrogens

At the beginning of the twentieth century, a relationship between the pituitary, testes and prostate gland had been established. American physician Charles Brenton Huggins found out that castration or estrogen administration led to glandular atrophy in men, which could be reversed by re-administration of androgen. In 1941 Huggins treated prostate cancer patients by androgen ablation with either castration or estrogen therapy; the beneficial effect of androgen ablation on metastatic prostate cancer was realised, for which he was awarded the Nobel Prize in Physiology or Medicine in 1966. It became evident that androgen ablation alone was insuffient to cure patients with advanced prostate cancer. In the late 1960s, the androgen receptor (AR) was discovered and characterized. Screening of chemical libraries for AR blockers led to the discovery of the first antiandrogen, cyproterone. An acetate group was then added to cyproterone and created cyproterone acetate. In the 1970s, the antiandrogen flutamide was discovered. In 1989 the United States Food and Drug Administration (FDA) approved it for use the treatment of prostate cancer. In 1995, bicalutamide was approved, and nilutamide followed a year later. ==Androgen receptor==

The AR belongs to the steroid receptor subfamily of the nuclear receptor superfamily. Its function is regulated by the binding of androgens, which initiates sequential conformation changes of the receptor that affects receptor-protein and receptor-DNA interactions. Endogenous androgens are mainly testosterone and DHT. AR is expressed in cells of a wide range of tissues, throughout the entire body, beyond primary and secondary sexual organs. The AR gene is more than 90kb long and codes for a protein of 919 amino acids. Only one AR gene has been identified in humans which is located on chromosome X. It comprises four main regions, see figure 1: • N-terminal domain (NTD) which serves a modulatory function. • DNA-binding domain (DBD) which recognises and binds to androgen response elements (ARE) in target gene sequence. • Ligand binding domain (LBD) which is responsible for ligand recognition and binding. • A small hinge region between the DBD and LBD. Two functions have been identified in AR that have critical roles in the regulation of target gene transactivation, the N-terminal activation function 1 (AF1) and the C-terminal activation function 2 (AF2). AF1 is ligand-independent and plays the primary role in target gene transactivation. The AF2 is a ligand-dependent and only shows limited function. Mechanism of action Unbound AR is mainly located in the cytoplasm, like a typical steroid receptor, and is associated with a complex of heat shock proteins (HSP) through interactions with LBD. Androgens, either agonists or antagonists, position themselves in the ligand-binding pocket (LBP) of the cytosolic AR and bind to the LBD, see figure 2. The AR goes through a series of conformational changes and HSP dissociate from AR. The transformed AR undergoes dimerisation, phosphorylation and translocates to the nucleus. The translocated receptor then binds to the androgen-response elements (ARE) on the promoter of the androgen responsive gene, a consensus sequence located either upstream or downstream of the transcription start site (TSS) of AR target genes. Recruitment of other transcription co-factors (including co-activators and co-repressors) and general transcriptional machinery further ensures the transactivation of AR-regulated gene expression. All these complicated processes are initiated by the ligand-induced conformational changes in the LBD. Ligand specific recruitment of coregulators might be crucial for the agonist or antagonist activity of AR ligands. Binding of DNA is also required for AR-regulated gene expression, also known as classic genomic gene function of AR. ==Development of antiandrogens==

Cyproterone is a steroidal antiandrogen that competitively inhibits the binding of testosterone or DHT to AR. Cyproterone binds to ARs that are expressed by prostate cancer cells as well as to the AR that are expressed in the hypothalamus and pituitary. Therefore, cyproterone blocks the negative feedback of androgens at the hypothalamic-pituitary level leading to increased luteinizing hormone (LH) serum levels. This rise in LH levels causes an increase in serum testosterone levels and ultimately diminishes the ability of cyproterone to compete for AR binding and to block androgenic stimulation. Nilutamide Nilutamide is a nitroaromatic hydantoin analog of flutamide, as seen in figure 5. Nilutamide is eliminated exclusively by metabolism, mainly by reduction of the aromatic nitro group. Although the hydrolysis of one of the carbonyl functions of the imidazolinedione was identified, it is much less susceptible to hepatic metabolism than the amide bond in hydroxuflutamide. This results in a longer half-life of nilutamide in humans of 2 days. Nevertheless, the nitro anion-free radical formed during nitro reduction could still be associated with hepatotoxicity in humans, especially when using relatively high dosage employed for androgen blockage. Nilutamide causes side-effects which limit its usage, such as pneumonitis and delayed adaption to darkness. Bicalutamide Bicalutamide is an arylpropionamide analog, seen in figure 6. It has replaced flutamide and nilutamide as the first choice antiandrogen for prostate cancer treatment. Bicalutamide is not as hepatotoxic as flutamide and nilutamide and has a longer half-life, of 6 days in humans, that allows once a day administration at lower dosage. Bicalutamide shares the amide bond structure with flutamide. Even so, the amide bond hydrolysis was discovered in rats, not in humans, which could explain the prolonged half-life of bicalutamide in humans. Bicalutamide has a cyano group at the para position instead of a nitro group like flutamide and nilutamide. This change in groups avoids the nitro reduction observed in nilutamide. Bicalutamide has a chiral carbon in its structure (labeled with an asterisk in figure 6), which is connected to the hydroxyl and methyl groups . It is therefore administered as a racemate. Post-approval investigation revealed that its antiandrogenic activity resides almost entirely in the (R)-enantiomer. (R)-bicalutamide has an almost fourfold higher affinity for the prostate AR than hydroxyflutamide and has a better side-effect profile compared to other antiandrogens. ==Structure and activity relationship==

Steroidal antiandrogens Cyproterone acetate is a 6-chloro-1,2-methylene derivative of 17α-acetoxyprogesterone. It shows major antiandrogenic activity together with androgenic activities. Cyproterone acetate displays high affinity for AR in rats which increases when the 1,2-methylene group is removed from the compound. If the chlorine atom is replaced by a methyl group the binding slightly decreases, whereas further removal of the C6 double bond modifies the binding kinetics, see figure 7. For hydroxyflutamide, a group of compounds that differed in the aromatic ring did not bind to the AR. This suggests that the bisubstitution in the hydroxyflutamide ring is essential for high AR binding affinity. It has also been demonstrated that hydroxyflutamide requires the strong hydrogen bond donor ability of the tertiary hydroxyl group and fixed conformers involved in intramolecular hydrogen binding, to bind effectively to AR. For bicalutamide, the antiandrogenic activities of sulfide and sulfone substitutions of the X-linkage were tested in vitro. The sulfides showed in most cases at least 2-fold higher binding affinity than corresponding sulfones. However, this relationship was reversed when the R3 group was NHSO2CH3, where the binding affinity of sulfone was 3-fold higher than that of sulfide. These results indicate that substituents of the B-ring largely determine the effect of the X-linkage in AR binding. Researchers have proposed that the tertiary hydroxyl group is involved in direct interaction with AR because when an acetyl group is introduced to that hydroxyl moiety, the receptor binding affinity greatly decreases. Nilutamide has very low affinity for AR when tested on castrated rat prostate. Modifications such as replacing the N3 atom with oxygen has little effect on affinity of the compound for prostate AR. By replacing the oxygen atom with a sulfur atom at the C2 position of the imidazole ring and adding butylalcohol to the N3 atom, the receptor binding and biological activity of the compound increases 100 times that of NSAAs. Also the compound does not bind to other steroid receptors. If a methyl group is changed for the butylalcohol group, the compound shows 3 and 10 times more antiandrogenic activity in vivo than bicalutamide and nilutamide, respectively. ==Antiandrogen withdrawal syndrome==

Antiandrogens that are currently on the market are particularly useful for the treatment of prostate cancer during the early stages. However, prostate cancer often progresses to a hormone-refractory state in which the cancer progresses in the presence of continued androgen ablation or antiandrogen therapy. This suggests that long term use of these antiandrogens during prostate cancer can lead to the development of androgen-independent prostate cancer cells or the ability of adrenal androgens to support tumor growth. This phenomenon is called antiandrogen withdrawal syndrome (AWS) and is one of the major drawbacks of existing antiandrogens. AWS is defined as tumor regression or symptomatic relief observed upon discontinuation of the antiandrogen therapy. The mechanism for this is not fully understood but current theories include alterations of the AR gene, coregulator proteins and/or signal transduction pathways. This antiandrogen resistance may also be linked to the relative weakness of current antiandrogens as they have an affinity 50 times or more lower than that of DHT for the AR. This may also explain why compensatory AR overexpression is often observed. ==Androgen receptor gene mutations==

AR gene mutations in the LBD that alter ligand specificity and/or functional activity exist and are thought to contribute to the conversion of some AR antagonists into agonists, which explains the paradoxical temporary improvement sometimes observed in patients when antiandrogen therapy is stopped. These mutations can have great effect on the antagonist activities of current small molecule antiandrogens and make them less efficient in blocking AR function via indirect modulation from inside of the LBP. Recent studies with circulating tumor cells, suggest that the mutation frequency is higher than previously assumed based on tumor biopsies. The T877A, W741L and W741C mutations are examples of known AR LBD mutations. The LNCaP prostate cancer cell line expresses AR with a T877A point mutation that causes proliferation in the presence of the antiandrogens hydroxyflutamide and cyproterone acetate. This mutation has also been discovered in patients with antiandrogen withdrawal syndrome being treated with these compounds. The W741L mutation generates additional space such that the sulfonyl-linked phenyl ring of bicalutamide is accommodated at the location of the missing indole ring of W741. In non-mutant AR, the presence of the W741 side chain probably forces bicalutamide to protrude out thus precluding the active position of H12 on the AR receptor. However, hydroxyflutamide worked as an antagonist for W741 mutant ARs. This concurs with the theory that flutamide and nilutamide antagonize AR through the mechanism of “passive antagonism”, as they are of a more modest size then bicalutamide. These drugs may therefore be effective as a second-line therapy for refractory prostate cancer previously treated with bicalutamide. ==Current status==

N-Terminal domain antagonists Antagonists of the N-terminal domain (NTD) of the AR have been proposed to overcome the limitations of current antiandrogens regarding mutant ARs, by directly blocking AR function from protein surface, outside of the LBP. This direct blockade is thought to provide a more efficient strategy to avoid or overcome abnormal AR action during AWS, as well as allowing for more flexibility in structural modification without the space limitations of the rigid LBP. The evidence presented was not sufficient to support the conclusion that sintokamide A directly inhibits the function of AR NTD, and the mechanism of action needs further investigation. Other small-molecule antiandrogens The development status of other small molecule antiandrogens undergoing research in 2011 can be seen in table 1. Natural antiandrogens Atraric acid and N-butylbenzenesulfonamide are natural compounds with antiandrogen properties which have been purified from the bark of the African tree Pygeum africanum, see figures 9 and 10. In vitro assays have shown them both to be selective AR agonists and that they inhibit proliferation of several prostate cancer cell lines. Atraric acid also hinders extracellular matrix invasion and both compounds are able to prevent androgen-induced nuclear translocation of the AR. More potent derivatives are currently being synthesized in hope of improving the pharmacological profile of these two compounds. ==See also==