Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3.

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 µM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties.

5α-reductase type 3 enzyme in benign and malignant prostate.

BACKGROUND: Currently available 5α-reductase inhibitors are not completely effective for treatment of benign prostate enlargement, prevention of prostate cancer (CaP), or treatment of advanced castration-recurrent (CR) CaP. We tested the hypothesis that a novel 5α-reductase, 5α-reductase-3, contributes to residual androgen metabolism, especially in CR-CaP. METHODS: A new protein with potential 5α-reducing activity was expressed in CHO-K1 cellsandTOP10 E. coli for characterization. Protein lysates and total mRNA were isolated from preclinical and clinical tissues. Androgen metabolism was assessed using androgen precursors and thin layer chromatography or liquid chromatography tandem mass spectrometry. RESULTS: The relative mRNA expression for the three 5α-reductase enzymes in clinical samples of CR-CaP was 5α-reductase-3 ≫ 5α-reductase-1> 5α-reductase-2. Recombinant 5α-reductase-3 protein incubations converted testosterone, 4-androstene-3,17-dione (androstenedione) and 4-pregnene-3,20-dione (progesterone) to dihydrotestosterone, 5α-androstan-3,17-dione, and 5α-pregnan-3,20-dione, respectively. 5α-Reduced androgen metabolites were measurable in lysates from androgen-stimulated (AS) CWR22 and CR-CWR22 tumors and clinical specimens of AS-CaP and CR-CaP pre-incubated with dutasteride (a bi-specific inhibitor of 5α-reductase-1 and 2). CONCLUSION: Human prostate tissues contain a third 5α-reductase that was inhibited poorly by dutasteride at high androgen substrate concentration in vitro, and it may promote DHT formation in vivo, through alternative androgen metabolism pathways when testosterone levels are low.

Preventive and therapeutic efficacy of finasteride and dutasteride in TRAMP mice.

BACKGROUND: The prostate cancer prevention trial (PCPT) and Reduction by dutasteride of Prostate Cancer Events (REDUCE) trial found that 5α-reductase (5αR) inhibitors finasteride and dutasteride respectively, decreased prostate cancer prevalence but also increased the incidence of high-grade tumors. 5αR2 is the main isoenzyme in normal prostate tissue; however, most prostate tumors have high 5αR1 and low 5αR2 expression. Because finasteride inhibits only 5αR2, we hypothesized that it would not be as efficacious in preventing prostate cancer development and/or progression in C57BL/6 TRAMP x FVB mice as dutasteride, which inhibits both 5αR1 and 5αR2. METHOD/PRINCIPAL FINDINGS: Six-week-old C57BL/6 TRAMP x FVB male mice were randomized to AIN93G control or pre- and post- finasteride and dutasteride diet (83.3 mg drug/kg diet) groups (n =30-33) that began at 6 and 12 weeks of age, respectively, and were terminated at 20 weeks of age. The pre- and post- finasteride and dutasteride groups were designed to test the preventive and therapeutic efficacy of the drugs, respectively. Final body weights, genitourinary tract weights, and genitourinary tract weights as percentage of body weights were significantly decreased in the Pre- and Post-dutasteride groups compared with the control. The Post-dutasteride group showed the greatest inhibition of prostatic intraepithelial neoplasia progression and prostate cancer development. Surprisingly, the Post-dutasteride group showed improved outcomes compared with the Pre-dutasteride group, which had increased incidence of high-grade carcinoma as the most common and most severe lesions in a majority of prostate lobes. Consistent with our hypothesis, we found little benefit from the finasteride diets, and they increased the incidence of high-grade carcinoma. CONCLUSION: Our findings have commonalities with previously reported PCPT, REDUCE, and the Reduction by dutasteride of Clinical Progression Events in Expectant Management (REDEEM) trial results. Our results may support the therapeutic use of dutasteride, but not finasteride, for therapeutic or preventive use.

New steroidal 17ß-carboxy derivatives present anti-5α-reductase activity and anti-proliferative effects in a human androgen-responsive prostate cancer cell line.

The androgens testosterone (T) and dihydrotestosterone (DHT), besides playing an important role in prostate development and growth, are also responsible for the development and progression of benign prostate hyperplasia (BPH) and prostate cancer. Therefore, the actions of these hormones can be antagonized by preventing the irreversible conversion of T into DHT by inhibiting 5α-reductase (5α-R). This has been a useful therapeutic approach for the referred diseases and can be achieved by using 5α-reductase inhibitors (RIs). Steroidal RIs, finasteride and dutasteride, are used in clinic for BPH treatment and were also proposed for chemoprevention of prostate cancer. Nevertheless, due to the increase in bone and muscle loss, impotency and occurrence of high-grade prostate tumours, it is important to seek for other potent and specific molecules with lower side effects. In the present work, we designed and synthesized steroids with the 3-keto-Δ(4) moiety in the A-ring, as in the 5α-R substrate T, and with carboxamide, carboxyester or carboxylic acid functions at the C-17ß position. The inhibitory 5α-R activity, in human prostate microsomes, as well as the anti-proliferative effects of the most potent compounds, in a human androgen-responsive prostate cancer cell line (LNCaP cells), were investigated. Our results showed that steroids 3, 4 and 5 are good RIs, which suggest that C-17ß lipophylic amides favour 5α-R inhibition. Moreover, these steroids induce a decrease in cell viability of stimulated LNCaP cells, in a 5α-R dependent-manner, similarly to finasteride.

Synthesis and characterization of 3ß-substituted amides of 17a-aza-D-homo- 4-androsten-17-one as potent 5α-reductase inhibitors and antimicrobial agents.

We herein report the synthesis of 3ß-substituted amides of 17a-aza-D-homo-4-androsten-17-one (11a-11r) from commercially available Diosgenin as the starting material. The structures of newly synthesized compounds were confirmed by IR, (1)H NMR, (13)C NMR and mass spectrometry. All the synthesized analogues were tested for their 5α- reductase inhibitory and antimicrobial activity, some of them exhibit moderate to potent activity comparable to the reference drugs. Among the synthesized derivatives the analogue (11r) 3ß-(indonlylbutanamido)-17a-aza-D-homo-4- androsten-17-one was found to be active against both 5α-reductase enzyme and microbial strains, whereas the analogue (11i) 3ß-(3,4-dimethoxy-benzamido)-17a-aza-D-homo-4-androsten-17-one was found to be the least active. The detailed 5α-reductase inhibitors and antimicrobial activities of the synthesized compounds were reported.

The implication of neuroactive steroids in Tourette's syndrome pathogenesis: A role for 5α-reductase?

Tourette's syndrome (TS) is a neurodevelopmental disorder characterised by recurring motor and phonic tics. The pathogenesis of TS is considered to reflect dysregulations in the signalling of dopamine (DA) and other neurotransmitters, which lead to excitation/inhibition imbalances in cortico-striato-thalamocortical circuits. The causes of these deficits may reflect complex gene × environment × sex (G × E × S) interactions; indeed, the disorder is markedly predominant in males, with a male-to-female prevalence ratio of approximately 4 : 1. Converging lines of evidence point to neuroactive steroids as being likely molecular candidates to account for G × E × S interactions in TS. Building on these premises, our group has begun examining the possibility that alterations in the steroid biosynthetic process may be directly implicated in TS pathophysiology; in particular, our research has focused on 5α-reductase (5αR), the enzyme catalysing the key rate-limiting step in the synthesis of pregnane and androstane neurosteroids. In clinical and preclinical studies, we found that 5αR inhibitors exerted marked anti-DAergic and tic-suppressing properties, suggesting a central role for this enzyme in TS pathogenesis. Based on these data, we hypothesise that enhancements in 5αR activity in early developmental stages may lead to an inappropriate activation of the 'backdoor' pathway for androgen synthesis from adrenarche until the end of puberty. We predict that the ensuing imbalances in steroid homeostasis may impair the signalling of DA and other neurotransmitters, ultimately resulting in the facilitation of tics and other behavioural abnormalities in TS.

5-α Reductase inhibitory effect and astringent activity of green apple rind extract on human keratinocytes and fibroblast cells.

Unripe green apples contain condensed tannins at 10 times higher levels than ripe apples. Tannin not only has strong antioxidant activity, but also an astringent property. In this study, we investigated the effects of green apple rind (GAR) extracts in reducing facial pores and sebum secretion. Among the GAR extracts, the 70% ethanol GAR extract showed the highest antioxidant activity and tannin content. Hence, it was further fractionated with different solvents. Among these rind solvent fractions, the ethyl acetate fraction of the extract (GAR-E) showed astringent activity. Additionally, it exhibited inhibitory effects on 5-α reductase, and induced type 1 collagen and involucrin synthesis. These results suggest that GAR-E can be applied in cosmetics to reduce facial pore size and sebum secretion.

Testosterone metabolites mediate its effects on myocardial damage induced by ischemia/reperfusion in male Wistar rats.

The role of testosterone in cardiovascular (CV) homeostasis is in controversy, and the exact effects of testosterone on the cardiovascular system remain poorly understood. Testosterone is metabolized by aromatase into 17ß-estradiol and by 5α-reductase into dihydrotestosterone (DHT). Thus, identification of these metabolites in the heart may help to explain the controversy regarding the cardiovascular effects of testosterone. We analyzed the expression patterns of these testosterone-metabolizing enzymes and assessed the effect of its enzymatic activity inhibition on ischemia (40 min)/reperfusion (4h, I/R) via the left anterior descendent coronary artery in intact and gonadectomized male rats. Myocardial damage was measured as percentage of infarcted area vs. area at risk. Aromatase and 5α-reductase protein expression was found in the left ventricle of intact and orchidectomized rats. Exogenous testosterone had no effect on I/R induced myocardial damage in intact male rats, meanwhile exogenous testosterone protects against I/R injury in orchidectomized rats. However, enzymatic inhibition of aromatase increased myocardial damage in the presence of testosterone, while enzymatic inhibition of 5α-reductase significantly decreased the level of myocardial damage. Our results also showed that sub-chronic inhibition of 5α-reductase resulted in myocardial protection in both groups. Furthermore, in orchidectomized and intact male rats IV treatment with DHT induces a significant increase in the myocardial damage induced by I/R. Thus, the effect of testosterone on cardiovascular pathophysiology could be related, at least in part to changes in the balance of testosterone 5α-reduction and aromatization.

In vivo and in vitro effect of androstene derivatives as 5α-reductase type 1 enzyme inhibitors.

The aim of these studies was to synthesize twelve ester derivatives of dehydroepiandrosterone with therapeutic potential. The effect of 1-12 was demonstrated in the flank organs of gonadectomized hamsters treated with testosterone and the synthesized steroids. In vitro studies were carried out determining the IC50 values for the inhibition of the activity of 5α-reductase type 1 and 2, which are present in rat liver and human prostate respectively. The binding of 1-12 to the androgen receptors (AR) was determined using rat's prostate cytosol. Steroids 1-12 containing different substituents in the phenyl group of the ester moiety in C-3 reduced the flank organs and inhibited the activity of 5α-R type 1; however only steroids 1 and 2 inhibited 5α-R type 2. 1-12 did not bind to the AR. The modification of one atom of the substituents in the phenyl group of the ester moiety in C-3 changed their biological potency (IC50).

5α-reductases in human physiology: an unfolding story.

OBJECTIVE: 5α-reductases are a family of isozymes expressed in a wide host of tissues including the central nervous system (CNS) and play a pivotal role in male sexual differentiation, development and physiology. METHODS: A comprehensive literature search from 1970 to 2011 was made through PubMed and the relevant information was summarized. RESULTS: 5α reductases convert testosterone, progesterone, deoxycorticosterone, aldosterone and corticosterone into their respective 5α-dihydro-derivatives, which serve as substrates for 3α-hydroxysteroid dehydrogenase enzymes. The latter transforms these 5α-reduced metabolites into a subclass of neuroactive steroid hormones with distinct physiological functions. The neuroactive steroid hormones modulate a multitude of functions in human physiology encompassing regulation of sexual differentiation, neuroprotection, memory enhancement, anxiety, sleep and stress, among others. In addition, 5α -reductase type 3 is also implicated in the N-glycosylation of proteins via formation of dolichol phosphate. The family of 5α-reductases was targeted for drug development to treat pathophysiological conditions, such as benign prostatic hyperplasia and androgenetic alopecia. While the clinical use of 5α-reductase inhibitors was well established, the scope and the magnitude of the adverse side effects of such drugs, especially on the CNS, is still unrecognized due to lack of knowledge of the various physiological functions of this family of enzymes, especially in the CNS. CONCLUSION: There is an urgent need to better understand the function of 5α-reductases and the role of neuroactive steroids in human physiology in order to minimize the potential adverse side effects of inhibitors targeting 5α-reductases to treat benign prostatic hyperplasia and androgenic alopecia.

Inhibitors of 5α-reductase-related side effects in patients seeking medical care for sexual dysfunction.

BACKGROUND: Despite their efficacy in the treatment of benign prostatic hyperplasia (BPH) the popularity of inhibitors of 5α-reductase (5ARI) is limited by their association with adverse sexual side effects. However, the real impact of 5ARI on sex hormones and sexual function is controversial. AIM: To investigate the role of 5ARI therapy on hormonal parameters and sexual function in men already complaining of sexual problems. MATERIALS AND METHODS: A consecutive series of 3837 men (mean age 63.5±12.8 yr) attending our outpatient clinic for sexual dysfunction was retrospectively studied. Several clinical, biochemical, and instrumental (penile color doppler ultrasound) factors were evaluated. RESULTS: Among the patients studied, 78.7% reported erectile dysfunction, 51.1% hypoactive sexual desire (HSD), 86.7% perceived reduced sleep-related erections (PR-SRE) and 19.1% premature ejaculation. The use of 5ARI was associated with an increased risk of HSD and PR-SR whereas no relationship was found with erectile dysfunction and ejaculation disturbances. Subjects using 5ARI also more frequently had gynecomastia along with reduced SHBG and higher calculated free testosterone levels. All these associations were confirmed in a case-control study comparing 5ARI users with age-body mass index-smoking status and total testosterone-matched controls. CONCLUSIONS: Our data indicates that use of 5ARI in men with sexual dysfunction does not significantly exacerbate pre-existing ejaculatory or erectile difficulties, but can further impair their sexual life by reducing sexual drive and spontaneous erection.

Synthesis and biological evaluation of novel unsaturated carboxysteroids as human 5α-reductase inhibitors: a legitimate approach.

In the present study, novel steroidal 17a-substituted 3-cyano-17a-aza-D-homo-3,5-androstadien-17-ones (12-19) and 17a-substituted 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acids (20-26) were synthesized from dehydroepiandrosterone acetate (6) along with 17-oxo-19-nor-3,5-androstadien-3-oic acid (30) through a multistep synthesis. Compounds were evaluated for their in vitro 5α-reductase inhibitory activity by measuring the conversion of [(3)H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity was also determined using rat prostate weighing method. Compounds 21-23 and 25 showed potent inhibition of 5α-reductase II enzyme with IC(50) values of 54.1 ± 9.5, 22.1 ± 2.4, 72.8 ± 2.3 and 26.5 ± 4.4 nM respectively as compared to Finasteride (30.3 nM) along with a significant (p < 0.05) reduction in rat prostate weight.

5α-Reductase, an enzyme regulating glucocorticoid action in the testis of Rhinella arenarum (Amphibia: Anura).

The reduction of A-ring of glucocorticoids to produce 5α-dihydro-derivatives by 5α-reductases has been considered as a pathway of irreversible inactivation. However, 5α-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5α-reductase (5α-Red) is able to transform corticosterone into 5α-dihydrocorticosterone. Furthermore, we studied the role of 5α-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5α-Red was assayed in subcellular fractions with [(3)H]corticosterone or [(3)H]testosterone as substrate. The enzyme localizes in microsomes and its optimal pH is between 7 and 8. The activity is not inhibited by finasteride. These results support the conclusion that toad 5α-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither K(m) nor V(max) for both corticosterone and testosterone were significantly different among reproductive periods. The K(m) value for testosterone was significantly higher than that for corticosterone, indicating that the C-21 steroid is the preferred substrate for the enzyme. Studies of the binding capacity of 5α-dihydrocorticosterone (5α-DHB) to the testicular GR show that 5α-DHB is able to displace the binding of [(3)H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5α-DHB were similar, 31.33±2.9 nM and 35.24±2.3 nM, respectively. In vitro experiments suggest that 5α-DHB is an agonist of toad testicular GR, decreasing the activity of the key enzyme for androgen synthesis, the cytochrome P450 17-hydroxylase, C17,20-lyase.

Inhibition of 5α-reductase activity in late pregnancy decreases gestational length and fecundity and impairs object memory and central progestogen milieu of juvenile rat offspring.

Psychological, physical and/or immune stressors during pregnancy are associated with negative birth outcomes, such as preterm birth and developmental abnormalities. In rodents, prenatal stressors can alter the expression of 5α-reductase enzymes in the brain and may influence cognitive function and anxiety-type behaviour in the offspring. Progesterone plays a critical role in maintaining gestation. In the present study, it was hypothesised that 5α-reduced progesterone metabolites influence birth outcomes and/or the cognitive and neuroendocrine function of the offspring. 5α-Reduced steroids were manipulated in pregnant Long-Evans rats via the administration of vehicle, the 5α-reduced, neuroactive metabolite of progesterone, 5α-pregnan-3α-ol-20-one (3α,5α-THP, allopregnanolone; 10 mg/kg/ml, s.c.), or the 5α-reductase inhibitor, finasteride (50 mg/kg/ml, s.c.), daily from gestational days 17-21. Compared to vehicle or 3α,5α-THP treatment, finasteride, significantly reduced the length of gestation and the number of pups per litter found in the dams' nests after parturition. The behaviour of the offspring in hippocampus-dependent tasks (i.e. object recognition, open field) was examined on post-natal days 28-30. Compared to vehicle-exposed controls, prenatal 3α,5α-THP treatment significantly increased motor behaviour in females compared to males, decreased progesterone content in the medial prefrontal cortex (mPFC) and diencephalon, increased 3α,5α-THP and 17ß-oestradiol content in the hippocampus, mPFC and diencephalon, and significantly increased serum corticosterone concentrations in males and females. Prenatal finasteride treatment significantly reduced object recognition, decreased hippocampal 3α,5α-THP content, increased progesterone concentration in the mPFC and diencephalon, and increased serum corticosterone concentration in female (but not male) juvenile offspring, compared to vehicle-exposed controls. Thus, inhibiting the formation of 5α-reduced steroids during late gestation in rats reduces gestational length, the number of viable pups per litter, and impairs cognitive and neuroendocrine function in the juvenile offspring.

Potential prostate cancer drug target: bioactivation of androstanediol by conversion to dihydrotestosterone.

High-affinity binding of dihydrotestosterone (DHT) to the androgen receptor (AR) initiates androgen-dependent gene activation, required for normal male sex development in utero, and contributes to prostate cancer development and progression in men. Under normal physiologic conditions, DHT is synthesized predominantly by 5α-reduction of testosterone, the major circulating androgen produced by the testis. During androgen deprivation therapy, intratumoral androgen production is sufficient for AR activation and prostate cancer growth, even though circulating testicular androgen levels are low. Recent studies indicate that the metabolism of 5α-androstane-3α, 17ß-diol by 17ß-hydroxysteroid dehydrogenase 6 in benign prostate and prostate cancer cells is a major biosynthetic pathway for intratumoral synthesis of DHT, which binds AR and initiates transactivation to promote prostate cancer growth during androgen deprivation therapy. Drugs that target the so-called backdoor pathway of DHT synthesis provide an opportunity to enhance clinical response to luteinizing-hormone-releasing hormone (LHRH) agonists or antagonists, AR antagonists, and inhibitors of 5α-reductase enzymes (finasteride or dutasteride), and other steroid metabolism enzyme inhibitors (ketoconazole or the recently available abiraterone acetate).