The clinical development of a 5 alpha-reductase inhibitor, finasteride.

Finasteride, a 4-aza steroid compound, is an orally active inhibitor of the 5 alpha-reductase enzyme. 5 alpha-Reductase is necessary for the metabolism of testosterone (T) to dihydrotestosterone (DHT) and is found in high levels only in certain tissues such as the prostate. Finasteride has been shown to markedly suppress serum DHT levels in man without lowering testosterone levels. In patients with benign prostate hyperplasia (BPH), finasteride was found to decrease prostate volume by a mean of 28% over a period of 6 months, without causing clinically significant adverse effects. DHT appears to be the primary androgen for prostatic growth. Selective inhibition of 5 alpha-reductase by finasteride may provide a novel approach to BPH therapy by reducing prostate size without affecting T-dependent processes such as fertility, muscle strength, and libido. The clinical development of finasteride for the treatment of benign prostate hyperplasia is reviewed.     

A kinetic analysis of the 5 alpha-reductases from human prostate and liver

A kinetic analysis of the 5 alpha-reductases from human liver and prostate is presented. Human prostatic 5 alpha-reductase follows an ordered sequential mechanism in which NADPH binds first followed by testosterone. The order of release of products is DHT followed by NADP+. The apparent Km of prostatic 5 alpha-reductase for testosterone is 0.0339 +/- 0.006 microM, while the apparent Km for NADPH is 2.52 +/- 0.65 microM. Human liver 5 alpha-reductase also follows a sequential mechanism. The apparent Km of the liver enzyme is 0.110 +/- 0.08 microM; the apparent Km for NADPH is 6.2 +/- 0.6 microM. The fact that both the liver and prostatic 5 alpha-reductases have a sequential kinetic mechanism rules out the possibility that the reduction of testosterone to dihydrotestosterone involves an electron transport system as previously proposed.     

Synthesis and bioactivity of new Finasteride conjugate

Finasteride is a synthetic 4-azasteroid compound that acts by inhibiting type II 5α-reductase, the enzyme that converts the androgen testosterone to 5α-dihydrotestosterone. It was approved by the US FDA for the treatment of benign prostatic hyperplasia and male pattern baldness. Here the acylation product of Finasteride C-18 amide N-polimod was synthesized by employing acylation reaction with polimod amide as a pivotal intermediate. The structure of the key intermediate and target molecule was confirmed by infrared spectrum, ¹H NMR and ¹³C NMR spectra and mass spectrum, and the inhibition of the steroid 5α-reductase and the rats’ benign prostatic hyperplasia by the new Finasteride conjugate and Finasteride was also determined. The inhibition of the Finasteride conjugate on 5α-reductase was stronger than that of Finasteride. Prostate hyperplasia of rats was reduced by Finasteride conjugate treatment similar to the Finasteride treatment. However, the Finasteride conjugate treated animals showed better viable condition than the Finasteride treated ones, suggesting the new compound may have improved toxicity profile than Finasteride.     

Selective non-steroidal inhibitors of 5 alpha-reductase type 1

The enzyme 5 alpha-reductase (5 alpha R) catalyses the reduction of testosterone (T) into the more potent androgen dihydrotestosterone (DHT). The abnormal production of DHT is associated to pathologies of the main target organs of this hormone: the prostate and the skin. Benign prostatic hyperplasia (BPH), prostate cancer, acne, androgenetic alopecia in men, and hirsutism in women appear related to the DHT production. Two isozymes of 5 alpha-reductase have been cloned, expressed and characterized (5 alpha R-1 and 5 alpha R-2). They share a poor homology, have different chromosomal localization, enzyme kinetic parameters, and tissue expression patterns. Since 5 alpha R-1 and 5 alpha R-2 are differently distributed in the androgen target organs, a different involvement of the two isozymes in the pathogenesis of prostate and skin disorders can be hypothesized. High interest has been paid to the synthesis of inhibitors of 5 alpha-reductase for the treatment of DHT related pathologies, and the selective inhibition of any single isozyme represents a great challenge for medical and pharmaceutical research in order to have more specific drugs. At present, no 5 alpha R-1 inhibitor is marketed for the treatment of 5 alpha R-1 related pathologies but pharmaceutical research is very active in this field. This paper will review the major classes of 5 alpha R inhibitors focusing in particular on non-steroidal inhibitors and on structural features that enhance the selectivity versus the type 1 isozyme. Biological tests to assess the inhibitory activity towards the two 5 alpha R isozymes will be also discussed.     

An overview on 5α-reductase inhibitors

Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5α-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5α-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5α-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5α-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5α-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5α-reductase have been covered.     

New 5alpha-reductase inhibitors: in vitro and in vivo effects

The enzyme 5alpha-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5alpha-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5alpha-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5alpha-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster's prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5alpha-reductase enzyme.     

4-pregnene-3-one-20 beta-carboxaldehyde: a potent inhibitor of 17 alpha-hydroxylase/C17,20-lyase and of 5 alpha-reductase.

The pregnene derivative, 4-pregnene-3-one-20 beta-carboxaldehyde (22-A) was evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and of 5 alpha-reductase in human prostatic homogenates. The effect of the compound in vivo was studied in adult male rats. The 22-A demonstrated potent and competitive inhibition of 17 alpha-hydroxylase and C17,20-lyase with Ki values 8.48 and 0.41 microM, respectively, significantly below the Km values for these two enzymes (33.75 and 4.55 microM). This compound also showed potent inhibition of 5 alpha-reductase with a Ki value of 15.6 nM (Km for this enzyme is 50 nM). By comparison, ketoconazole, a currently studied 17 alpha-hydroxylase/C17,20-lyase inhibitor for the treatment of prostatic cancer, showed less potent inhibition of 17 alpha-hydroxylase (Ki 39.5 microM) and C17,20-lyase (Ki 3.6 microM) and did not inhibit 5 alpha-reductase. Progesterone which has been reported to inhibit the 17 alpha-hydroxylase/C17,20-lyase, did not significantly reduce the production of testosterone by rat testes in vitro in comparison to controls, while the same concentration of 22-A demonstrated a 42% reduction of testosterone biosynthesis. When the adult male rats were injected s.c. with 22-A at 50 mg/day/kg for a 2 week period, the testosterone concentrations in the rat sera were significantly lower than control values (P less than 0.05), whereas serum corticosterone levels did not change. These results suggest that 22-A is a selective potent inhibitor for 17 alpha-hydroxylase and C17,20-lyase, but is more potent for the C17,20-lyase. The compound also inhibits 5 alpha-reductase, and therefore may reduce biosynthesis of testosterone and dihydrotestosterone effectively. Thus, 22-A may be useful in the treatment of problems associated with the androgen excess and prostatic cancer.     

Finasteride induced depression: a prospective study

Background  

Finasteride is a competitive inhibitor of 5 alpha-reductase enzyme, and is used for treatment of benign prostatic hyperplasia and androgenetic alopecia. Animal studies have shown that finasteride might induce behavioral changes. Additionally, some cases of finasteride-induced depression have been reported in humans. The purpose of this study was to examine whether depressive symptoms or anxiety might be induced by finasteride administration.     

Retinoic acid (RA): an inhibitor of 5 alpha-reductase in human prostatic cancer cells

The effect of retinoic acid (RA) on testosterone metabolism was examined in a prostatic cancer cell line of human origin, PC-3. In cells growing as monolayers as well as in cell homogenates RA causes a dose-dependent inhibition of the 5 alpha-reductase activity, thus preventing the conversion of testosterone into its hormonally active metabolite, dihydrotestosterone. Fifty per cent inhibition of the enzyme activity occurred at an RA concentration of 2 x 10(-5)M. The pattern of inhibition was that of a non-competitive inhibitor. However, when incubations were performed in the presence of varying amounts of NADPH, it turned out that RA exerts its effect by competitive inhibition of the cofactor action. Although the severe toxicity of RA precludes its systemic use as a 5 alpha-reductase inhibitory drug in humans, the possible anti-androgenic effect of other, less toxic, retinoids should be investigated.     

Non-michaelian behavior of 5 alpha-reductase in human prostate

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.     

5 alpha-reductase-catalyzed conversion of testosterone to dihydrotestosterone is increased in prostatic adenocarcinoma cells: suppression by 15-lipoxygenase metabolites of gamma-linolenic and eicosapentaenoic acids

Although the androgens, testosterone (T) and its highly active metabolite dihydrotestosterone (DHT) play a role in the development and progression of prostate cancer, the mechanism(s) are unclear. Furthermore, 5 alpha-reductase which catalyze the conversion of T to DHT, has been a target of manipulation in the treatment of prostatic cancer, hence synthetic 5 alpha-reductase activity inhibitors have shown therapeutic promise. To demonstrate that nutrients derived from dietary sources can exert similar therapeutic promise, this study was designed using benign hyperplastic cells (BHC) and malignant tumorigenic cells (MTC) derived from Lobund-Wistar (L-W) rat model of prostatic adenocarcinoma to test the effects of gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and their 15-lipoxygenase metabolites on cellular 5 alpha-reductase activity. Our data revealed: (i) that incubation of MTC with [3H]-T resulted in marked conversion to [3H]-DHT when compared to similar incubation with BHC; (ii) that DHT-enhanced activity of 5 alpha-reductase was inhibited 80% by 15S-hydroxyeicosatrienoic acid, the 15-lipoxygenase metabolite of GLA, when compared to 55% by 15S-hydroxyeicosapentaenoic acid, the 15-lipoxygenase metabolite of EPA; and (iii) that their precursor fatty acids, respectively, exerted moderate inhibition. Taken together, the study underscores the biological importance of 15-lipoxygenase metabolites of polyunsaturated fatty acids (PUFAs) in androgen metabolism.     

Effect of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium crustosum broths

The conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT) has been demonstrated in Penicillium crustosum broth obtained from fermented pistachios, lemons and corn tortillas. Furthermore, the presence of 5alpha-reductase enzyme, which is responsible for this conversion, has been established by electrophoretical techniques in these cultures.5alpha-Reductase enzyme is also present in animal and human androgen-dependent tissues as well as in prostate and seminal vesicles. The increase of the conversion of T to DHT in prostate gland, has been related to some illnesses such as benign prostate hyperplasia and prostate cancer. Furthermore, treatment with 5alpha-reductase inhibitors such as finasteride reduces the prostate growth. These data have stimulated research for the synthesis of new molecules with antiandrogenic activity, whose biological effect needs to be demonstrated.The purpose of this study is to determine the inhibition pattern of 5alpha-reductase in P. crustosum by finasteride and the new steroidal compound PM-9. K(m) and V(max) values for T, were determined in the broths by Lineweaver-Burk plots using different testosterone concentrations. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk using different concentrations of T and inhibitors. Results show that finasteride and PM-9 inhibit 5alpha-reductase present in the broth in a competitive manner.     

Solubilization and partial characterization of rat epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase).

Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 x 10(-7) - 4.52 x 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.     

The influence of 4-hydroxy-4-androstene-3,17-dione on androgen metabolism and action in cultured human foreskin fibroblasts

4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5 alpha-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent Km of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent Ki of 2.7 nM. When 5 alpha-reductase activity was determined in the presence of 200 nM [3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 microM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, androstenedione. The influence of 4-OHA on 5 alpha-reductase activity and androgen receptor binding is minimal.     

Tissue distribution and kinetic characteristics of rat steroid 5 alpha-reductase isozymes. Evidence for distinct physiological functions.

The enzyme steroid 5 alpha-reductase (5 alpha-reductase) catalyzes the reduction of delta 4,5 double bonds in a variety of substrates and is thought to play both catabolic and anabolic roles in steroid hormone metabolism. Here, we describe the isolation and characterization of a cDNA encoding the rat type 2 isozyme of 5 alpha-reductase and compare the kinetic properties and tissue-specific expression patterns of this isozyme with those of the type 1 isozyme. The type 2 isozyme has apparent Km values in the nanomolar range for steroid substrates, whereas the type 1 isozyme has micromolar affinities. The isozymes differ in their inhibition by various 4-azasteroids with the type 2 isozyme showing exquisite sensitivity (Ki = 40 pM) to 21,21-pentamethylene-4-aza-5 alpha-pregn-1-ene-3,20-dione. Messenger RNAs encoding the type 2 isozyme are more abundant than type 1 mRNAs in most male reproductive tissues, whereas the type 1 mRNAs predominate in peripheral tissues. Both 5 alpha-reductase mRNAs are more efficiently induced by dihydrotestosterone than by testosterone in the regenerating prostate. The differences in substrate affinities and tissue distributions of the 5 alpha-reductase isozymes suggest that type 2 plays an anabolic role and type 1 a catabolic role in the metabolism of androgens and other steroid hormones.     

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3 alpha(beta)-hydroxysteroid:NAD(P)+ oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of Ki for their inhibition of this enzyme are similar to that of the Km for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5 alpha-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD.     

Tissue and serum levels of principal androgens in benign prostatic hyperplasia and prostate cancer

Androgens are considered to play a substantial role in pathogenesis of both benign prostatic hyperplasia (BPH) and prostate cancer. The importance of determination of androgen levels in tissue and serum for cancer progression and prognosis has been poorly understood. The aim of study was to find out hormonal differences in both diseases, their correlations between intraprostatic and serum levels and predicted value of their investigation. Testosterone, dihydrotestosterone, androstenedione and also epitestosterone were determined in prostate tissue from 57 patients who underwent transvesical prostatectomy for BPH and 121 patients after radical prostatectomy for prostate cancer. In 75 subjects with cancer and 51 with BPH the serum samples were analyzed for testosterone, dihydrotestosterone and SHBG. Significantly higher intraprostatic androgen concentrations, i.e. 8.85+/-6.77 versus 6.44+/-6.43 pmol/g, p<0.01 for dihydrotestosterone, and 4.61+/-7.02 versus 3.44+/-4.53 pmol/g, p<0.05 for testosterone, respectively, were found in patients with prostate cancer than in BPH. Higher levels in cancer tissue were found also for epitestosterone. However, no differences were found in serum levels. Highly significant correlations occurred between all pairs of intraprostatic androgens and also epitestosterone as well as between serum testosterone and dihydrotestosterone (p<0.001) in both BPH and cancer groups. Correlation was not found between corresponding tissue and serum testosterone and dihydrotestosterone, either in benign or cancer samples. The results point to importance of intraprostatic hormone levels for evaluation of androgen status of patients, contrasting to a low value of serum hormone measurement.     

Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: elucidation of the kinetic mechanism

A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.     

Structure-activity relationship for inhibition of 5alpha-reductase by triterpenoids isolated from Ganoderma lucidum

In humans, 5alpha-reductase is involved in the development of benign prostatic hyperplasia. Triterpenoids isolated from ethanol extracts of Ganoderma lucidum (Fr.) Krast (Ganodermataceae) inhibited 5alpha-reductase activity. The presence of the C-3 carbonyl group and of the C-26-alpha,beta-unsaturated carbonyl group was characteristic of almost all inhibitors isolated from G. lucidum.