Alpha-reductase Isozymes Research Articles

MP83-14 GENOMIC EXPRESSION EVIDENCE FOR ANDROGEN RECEPTOR AXIS ACTIVATION IN UROTHELIAL CARCINOMA: DATA FROM THE CANCER GENOME ATLAS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-14 GENOMIC EXPRESSION EVIDENCE FOR ANDROGEN RECEPTOR AXIS ACTIVATION IN UROTHELIAL CARCINOMA: DATA FROM THE CANCER GENOME ATLAS Edwin E. Morales, Stephen B. Williams, Jinesh G. Goodwin, Debasish Sundi, Carolyn L. Smith, David J. McConkey, and Ashish M. Kamat Edwin E. MoralesEdwin E. Morales More articles by this author , Stephen B. WilliamsStephen B. Williams More articles by this author , Jinesh G. GoodwinJinesh G. Goodwin More articles by this author , Debasish SundiDebasish Sundi More articles by this author , Carolyn L. SmithCarolyn L. Smith More articles by this author , David J. McConkeyDavid J. McConkey More articles by this author , and Ashish M. KamatAshish M. Kamat More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2196AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent investigations have implicated the androgen receptor in the oncogenesis and progression of urothelial carcinoma. Current evidence exists that inhibition of dihydrotestosterone (DHT) production decreases the risk of bladder cancer by nearly a third in a large North American cancer screening cohort. The precise biological mechanism of DHT inhibition and reduced bladder carcinogenesis remains to be elucidated. We sought to analyze the bladder database of the Cancer Genome Atlas (TCGA) for androgen gene signature enrichment, and for expression of downstream target genes of the androgen receptor using heat mapping and genome set enrichment analysis. METHODS The TCGA is a multicenter study that examined 11,000 patient tumors as well as normal tissues to generate a genetic atlas of 33 distinct cancer types. The bladder TCGA was queried for expression of androgen axis gene expression signatures using GENE-E software (Broad Institute, Cambridge MA). Using this data, gene expression patterns were analyzed and using hierarchical clustering, matched to existing TCGA basal and luminal tumor subtypes. RESULTS Expression of the 5-alpha reductase isozymes SRD5A1, 2, and 3 (the inhibitory target of finasteride) was confirmed using the 128 tumor TCGA database and clustered into basal and luminal TCGA subtypes, with Clusters 1 and 2 (luminal or “epithelial-like”) expressing SRD5A2 and 3 while basal and “p-53 like” tumors expressed the more typical prostatic SRD5A1. In addition, significant expression of steroidal pathway synthesis and androgen/estrogen axes genes were observed, again clustered by TCGA subtype. Validation of these preliminary findings are underway. CONCLUSIONS Gene expression data suggest that urothelial cancer of the bladder exhibits activation of androgen synthesis and androgen receptor cross-talk pathways along with expression of the enzyme 5-alpha reductase. Expression appears to segregate by TCGA subtype which may be important in future clinical trials for subtype-specific antitumor therapies. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1086 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Edwin E. Morales More articles by this author Stephen B. Williams More articles by this author Jinesh G. Goodwin More articles by this author Debasish Sundi More articles by this author Carolyn L. Smith More articles by this author David J. McConkey More articles by this author Ashish M. Kamat More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Report on the two‐phase public consultation on the draft EFSA scientific opinion on bisphenol A (BPA)

Report on the two‐phase public consultation on the draft EFSA scientific opinion on bisphenol A (BPA)

Differential expression of 5-alpha reductase isozymes in the prostate and its clinical implications

The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone (T) and dihydrotestosterone (DHT). T is converted to DHT by 5-alpha reductase (5-AR) isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors (5-ARI) are commonly used for the treatment of benign prostatic hyperplasia (BPH) and were once promoted as chemopreventive agents for prostate cancer (PCa). This review discusses the role of the differential expression of 5-AR in the normal development of the human prostate and in the pathogenesis and progression of BPH and PCa.

The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family.

Consequences of Switching 5α-Reductase Inhibitors on Prostate Specific Antigen Velocity

The 5alpha-reductase inhibitors improve urinary symptoms related to benign prostatic hyperplasia, deter benign prostatic hyperplasia progression and provide prostate cancer chemoprevention. Currently there are a number of 5alpha-reductase inhibitor formularies, including Proscar, generic finasteride and dutasteride. While all formularies decrease serum prostate specific antigen (a proxy for prostate volume), they may not accomplish this to the same degree, which may have dramatic effects on prostate specific antigen kinetics in men changing 5alpha-reductase inhibitor formularies. We examined prostate specific antigen velocity after changes in 5alpha-reductase inhibitor formularies. We identified patients treated with 2 or more 5alpha-reductase inhibitor formularies who had sufficient prostate specific antigen values to calculate prostate specific antigen velocity during each 5alpha-reductase inhibitor treatment. Patient data were grouped depending on the formularies received. Statistical analysis was done to compare prostate specific antigen velocity at various time points while on different 5alpha-reductase inhibitors. Eight men changed from dutasteride to generic finasteride (group 1), 21 changed from dutasteride to Proscar (group 2), 49 changed from Proscar to dutasteride (group 3) and 77 changed from Proscar to generic finasteride (group 4). We noted a significant increase in prostate specific antigen velocity in groups 1 and 2 (p <0.05), and 4 (p <0.005). The increase was greater than 0.35 ng/ml per year, the common cutoff for prostate biopsy recommendations, in more than a third of patients. Results confirm that changing 5alpha-reductase inhibitors drugs can be associated with a clinically significant change in prostate specific antigen velocity. These prostate specific antigen velocity changes could place patients at risk for unnecessary prostate biopsy. Additional prospective studies are warranted.

5α‐Reductase Isozymes and Androgen Actions in the Prostate

Androgens acting via the androgen receptor play critical roles in prostate development, growth, and pathogenesis. There are two potent androgens, testosterone and dihydrotestosterone (DHT), in humans and mammals. DHT is converted from testosterone by 5alpha-reductase isozymes. Two 5alpha-reductase isozymes have been identified. Although both isozymes are expressed, 5alpha-reductase-2 is the predominant isozyme in the human prostate. Mutations in 5alpha-reductase-2 gene cause the 5alpha-reductase-2 deficiency syndrome. Affected 46, XY individuals have a small, nonpalpable, and rudimentary prostate in adulthood. Neither benign prostate hyperplasia (BPH) nor prostate cancer has been reported in these patients. The prostate is small in animals with 5alpha-reductase-2 gene knockout or treated with specific 5alpha-reductase inhibitors. 5alpha-reductase isozymes are molecular targets for the prevention and treatment of BPH and prostate cancer. Moreover, androgen actions on prostate gene expression and cell growth are directly modulated by estrogen receptor ligands via protein-protein interactions. The studies of 5alpha-reductases and androgen actions highlight the importance of 5alpha-reductase isozymes in male sexual differentiation and prostate physiology and pathophysiology.

Effects of dutasteride on the expression of genes related to androgen metabolism and related pathway in human prostate cancer cell lines

Androgens play an important role in controlling the growth of the normal prostate gland and in the pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5alpha-reductase isozymes, type I and II, is the major androgen in the prostate cells. The aim of this study is to investigate the cellular and molecular effects of dutasteride, a potent inhibitor of 5alpha-reductase type I and type II, in androgen-responsive (LNCaP) and androgen-unresponsive (DU145) human prostate cancer(PCa) cell lines. The expression pattern of 190 genes, selected on the basis of their proved or potential role in prostate cancerogenesis related to androgen signalling, were analysed using a low density home-made oligoarray (AndroChip 2). Our results show that dutasteride reduces cell viability and cell proliferation in both cell lines tested. AndroChip 2 gene signature identified in LNCaP a total of 11 genes differentially expressed (FC >or= +/-1.5). Eight of these genes, were overexpressed and three were underexpressed. Overexpressed genes included genes encoding for proteins involved in biosynthesis and metabolism of androgen (HSD17B1;HSD17B3;CYP11B2), androgen receptor and androgen receptor co-regulators (AR;CCND1), and signal transduction(ERBB2; V-CAM; SOS1) whereas, underexpressed genes (KLK3; KLK2; DHCR24) were androgen-regulated genes (ARGs). No differentially expressed genes were scored in DU145. Microarray data were confirmed by quantitative real-time PCR assay (QRT-PCR). These data offer a selective genomic signature for dutasteride treatment in prostate epithelial cells and provide important insights in prostate cancer pathophysiology.

Effects of Dihydrotestosterone on Brain mRNA Levels of Steroid 5α-Reductase Isozymes in Early Postnatal Life of Rat

We studied the expression of type 1 (5alpha-R1) and type 2 (5alpha-R2) 5alpha-reductase isozymes (5alpha-R) and their regulation by dihydrotestosterone (DHT) in the prefrontal cortex of male and female rats during postnatal sexual differentiation of the central nervous system (CNS), using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis. We found a higher expression of 5alpha-R2, which is considered a masculinizing enzyme, in the female versus male CNS, and observed sexual dimorphism in the regulation of both 5alpha-R isozymes by DHT. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.

Prooxidant and antioxidant action of 4-(4-phenoxybenzoyl)benzoic acid derivatives.

4-(4-Phenoxybenzoyl)benzoic acid derivatives (PBADs) were found to inhibit rat and human alpha-reductase isozymes 1 and 2 in vitro. Chemiluminescence (CL), electron spin resonance, spin trapping techniques, and spectrophotometry were used to examine the effect of PBADs on reactive oxygen species (superoxide radical, O(2)(.-); hydroxyl radical, HO(*); singlet oxygen, (1)O(2)) generating systems. All test compounds at a concentration of 0.5 mM enhanced the CL from O(2)(.-) up to fivefold, which was recorded as the light sums during 1 min. At 0.38 mM PBAD enhanced production of HO(*) from H(2)O(2) in the presence of Co(II) up to 90%, as measured by a deoxyribose assay. Using the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide, it was found that the amplitude of the signal arising from the Fenton-like reaction [Co(II)/H(2)O(2)] was significantly diminished by the test compounds. The compounds also inhibited the (1)O(2) dependent 2,2,6,6-tetramethylpiperidine-N-oxide radical, which is generated in the acetonitrile/H(2)O(2) system. The measured rate constants of (1)O(2)-dimol quenching by PBAD were in the range of (0.8-2.6) x 10(8) M(-1) s(-1). The interaction between PBAD and (1)O(2) was also checked using a spectrophotometry method based on bleaching of p-nitrosodimethylaniline. These results indicate that PBAD may directly scavenge HO(*) and (1)O(2), but not O(2)(.-). However, the compounds that were examined had prooxidant ability under some reaction conditions.

Quantitation of mRNA levels of steroid 5alpha-reductase isozymes: A method that combines one-step reverse transcription-polymerase chain reaction and separation by capillary electrophoresis.

We developed an accurate, rapid, and modestly labor-intensive method to precisely quantitate mRNA species by one-step reverse transcription-polymerase chain reaction (RT-PCR). This approach combines the high specificity of quantitative competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were coamplified in a single reaction in which the same primers are used. The 5' forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). The ratio of fluorescence intensity between amplified products of the target cDNA and the competitive DNA was determined quantitatively after separation by CE and fluorescence analysis. Using this method, we have been able to precisely quantify the mean amount of steroid 5alpha-reductase (5alpha-R) isozyme mRNA levels in ventral prostate of the rat, detecting 10-fold difference for 5alpha-R1 and 50-fold difference for 5alpha-R2, respectively, in comparison with our previously reported two-step method. Because the competitive RT-PCR presented in this paper enables a more efficient quantitative determination of mRNAs, low-level gene expression could be quantified.

Androgen-induced prostate-specific antigen gene expression is mediated via dihydrotestosterone in LNCaP cells.

Prostate cancer is a leading cause of cancer death in American males. Androgens play an essential role in prostate development, growth and pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5alpha-reductase isozymes, type 1 and 2, is the major androgen in the prostate cells. Thus, 5alpha-reductase(s) are critical in determining androgen activity in the prostate. However, it is unclear in prostate tumor cells whether 1 or 2 5alpha-reductase isozymes are expressed and whether they are functionally important. In the present report, we studied the importance of 5alpha-reductase isozymes in the androgen induction of prostate-specific antigen (PSA) gene expression in LNCaP prostatic tumor cells. Treatment with either testosterone or DHT in LNCaP cells produced dose- and time-dependent increases in PSA levels in the cell media and in PSA messenger RNA (mRNA) levels in the cells. However, testosterone-induced but not DHT-induced PSA gene expression was significantly inhibited by finasteride, a 5alpha-reductase inhibitor, in a dose-dependent manner. Furthermore, we demonstrated for the first time that both 5alpha-reductase-1 and 5alpha-reductase-2 mRNAs were expressed in LNCaP cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR Southern blot analysis. These results suggest that both 5alpha-reductase isozymes are present and functionally important in prostatic tumor LNCaP cells and that DHT is a major mediator of androgen induction of PSA gene expression in these cells.

Cyclohex‐1‐ene Carboxylic Acids: Synthesis and Biological Evaluation of Novel Inhibitors of Human 5α Reductase.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Cyclohex-1-ene carboxylic acids: synthesis and biological evaluation of novel inhibitors of human 5 alpha reductase.

In search of novel nonsteroidal mimics of steroidal inhibitors of 5 alpha reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acids 1-5 were synthesized with different substituents in para position of the phenyl ring (1: N, N-diisopropylcarbamoyl, 2: phenyl, 3: phenoxy, 4: benzoyl, and 5: benzyl). The principal synthetic approach for the desired compounds consisted of a Wittig olefination between 1, 4-dioxaspiro [4.5]-decane-8-carbaldehyde (4g and the appropriate phosphonium salts. The compounds were tested for inhibition of human 5 alpha reductase isozymes 1 and 2 using DU 145 cells and preparations from prostatic tissue, respectively. They turned out to be good inhibitors of the prostatic isozyme 2 with compound 1 being the most potent one (IC(50) = 760 nM). Isozyme 1 was only slightly inhibited. It is concluded that the novel structures are appropriate for being further optimized, aiming at the development of a novel drug for the treatment of benign prostatic hyperplasia.

Human osteoblast-like cells express predominantly steroid 5alpha-reductase type 1.

In previous studies we established that human bone and human osteoblast-like cells (hOB cells) cultured from bone express 5alpha-reductase (5alpha-R) activity, as demonstrated by the conversion of testosterone and androstenedione to their corresponding 5alpha-reduced metabolites, 5alpha-dihydrotestosterone (DHT) and 5alpha-androstanedione. Two 5alpha-R isozymes (types 1 and 2) have been identified in various tissues. As their nature in bone is unknown, we investigated which isozymes were expressed in first passage hOB cells cultured from bone specimens obtained from six donors (five women and one man). For comparison, 5alpha-reductase isozyme expression in genital skin fibroblasts cultured from foreskin of three males was determined. Pharmacological and biochemical studies using selective inhibitors of the 5alpha-R isozymes were performed, and gene expression was assessed by RT-PCR. In hOB cells, LY191704, a potent nonsteroidal selective inhibitor of 5alpha-R type 1, and the 4-azasteroid 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one (a dual inhibitor of 5alpha-R types 1 and 2) inhibited 5alpha-R activity with a 50% inhibitory concentration (IC(50)) of approximately 4 nM. Finasteride, a selective inhibitor of 5alpha-R type 2, blocked 5alpha-R activity with an IC(50) of approximately 60 nM. The IC(50) of progesterone, a physiological substrate for 5alpha-R, was approximately 200 nM. In genital skin fibroblasts, LY191704 inhibited 5alpha-R with an IC(50) of more than 5000 nM, whereas finasteride and 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one effectively inhibited 5alpha-R with IC(50) of approximately 4 nM. Experiments to determine 5alpha-reductase activity in homogenates of hOB cells as a function of pH showed very low activity at pH 5.5, but a broad shoulder of activity from pH 6.0-9.0, which was not inhibited by finasteride, but was nearly completely blocked by LY191704. RT-PCR revealed that 5alpha-R type 1 and 2 mRNAs were expressed in both bone and genital skin fibroblasts. Based on our pharmacological and biochemical studies, it appears that 5alpha-R activity in hOB cells is catalyzed predominantly by the type 1 rather than the type 2 isozyme. This expression pattern is in contrast to that in genital skin fibroblasts, where the activity of the type 2 isozyme prevails. As in most androgen target tissues DHT is biologically more active as an androgen than testosterone, DHT is formed in bone by 5alpha-R type 1 action from circulating testosterone, and bone cells also express the androgen receptor, local DHT production may play a physiological role in human bone homeostasis.

Synthesis and evaluation of 2'-substituted 4-(4'-carboxy- or 4'-carboxymethylbenzylidene)-N-acylpiperidines: highly potent and in vivo active steroid 5alpha-reductase type 2 inhibitors.

Sixteen compounds derived from N-acyl-4-benzylidenepiperidine-4'-carboxylic acids were synthesized and evaluated for inhibition of rat and human steroid 5alpha-reductase isozymes types 1 and 2. In the dicyclohexylacetyl series, fluorination in the 2-position of the benzene nucleus (15), exchange of the carboxy group by a carboxymethyl moiety (20), and combination of both structural modifications (25) led to highly active inhibitors of the human type 2 isozyme (IC(50) values: 15, 11 nM; 20, 6 nM; 25, 7 nM; finasteride, 5 nM). In vivo all compounds tested markedly reduced the prostate weights in castrated testosterone-treated rats. Oral activity was shown for compound 7. From the finding that compound 15 is active in the rat, although it is a rather poor inhibitor of the rat enzyme and is a strong inhibitor of the human enzyme, it is concluded that it should be highly potent in men.

Synthesis and Biological Evaluation of 4-(4-(Alkyl-and Phenylaminocarbonyl)benzoyl)benzoic Acid Derivatives as Non-steroidal Inhibitors of Steroid 5alpha-Reductase Isozymes 1 and 2.

The synthesis and biological evaluation of 4-(4-(alkyl- and phenylaminocarbonyl)-benzoyl)benzoic acids (4a-4d) as non-steroidal inhibitors of steroid 5alpha-reductase are described. The compounds were tested in vitro for inhibitory activity toward rat and human 5alpha-reductase isozymes 1 and 2 at a concentration of 10 &mgr;M.The most active inhibitor for the human type 2 isozyme was 4-(4-(phenylaminocarbonyl)benzoyl) benzoic acid, compound 4c (IC (50) = 0.82 &mgr;M).

Synthesis and biological evaluation of 4-(4-(alkyl- and phenylaminocarbonyl)benzoyl)benzoic acid derivatives as non-steroidal inhibitors of steroid 5 alpha-reductase isozymes 1 and 2.

The synthesis and biological evaluation of 4-(4-(alkyl- and phenylaminocarbonyl)benzoyl)benzoic acids (4a-4d) as non-steroidal inhibitors of steroid 5 alpha-reductase are described. The compounds were tested in vitro for inhibitory activity toward rat and human 5 alpha-reductase isozymes 1 and 2 at a concentration of 10 microM. The most active inhibitor for the human type 2 isozyme was 4-(4-(phenylaminocarbonyl)benzoyl)benzoic acid, compound 4c (IC50 = 0.82 microM).

5alpha-reductases in human breast carcinoma: possible modulator of in situ androgenic actions.

The expression of 5alpha-reductase types 1 and 2 was examined in human breast carcinoma using immunohistochemistry and RT-PCR. Immunoreactivity for 5alpha-reductase isozymes was also correlated with various clinicopathological parameters to examine possible local regulatory mechanisms of sex steroids, including progesterone and androgens, in human breast carcinoma tissues. Immunoreactivity for 5alpha-reductase type 1 was detected in the cytoplasm and possibly in the nuclear membrane of tumor cells in 35 of 60 invasive ductal carcinomas (58%), and type 2 signal was detected in 9 of these 60 cases (15%). The results from RT-PCR (n = 8) were consistent with those from immunohistochemistry. A significant positive correlation was detected between 5alpha-reductase type 1 immunoreactivity and androgen and progesterone receptor A or B labeling indexes, and immunoreactivities of 5alpha-reductase type 2, 17beta-hydroxysteroid dehydrogenase type 5, or 3beta-hydroxysteroid dehydrogenase, which recognizes both types I and II. An inverse correlation was detected between 5alpha-reductase type 1 immunoreactivity and tumor size, histological grade, or Ki-67 labeling index. 5alpha-Reductase type 2 immunoreactivity was significantly correlated with 17beta-hydroxysteroid dehydrogenase type 5 immunoreactivity, but not with other parameters. This study suggests that 5alpha-reductase type 1 is mainly expressed in human breast carcinoma, which may play an important role in the in situ production and actions of the potent androgen, 5alpha-dihydrotestosterone, including inhibition of cancer cell proliferation, in hormone-dependent human breast carcinoma.

Synthesis and Evaluation of Novel Steroidal Oxime Inhibitors of P450 17 (17α-Hydroxylase/C17−20-Lyase) and 5α-Reductase Types 1 and 2

17 alpha-Hydroxylase/C17-20-lyase (P450 17, CYP 17) and 5 alpha-reductase are the key enzymes in androgen biosynthesis and targets for the treatment of prostate cancer and benign prostatic hyperplasia. In the search of inhibitors for both enzymes, 23 pregnenolone- or progesterone-based steroids were synthesized bearing an oxime group connected directly or via a spacer to the steroidal D-ring. Tested for inhibition of human and rat P450 17, some pregnenolone (9, 11, 14) and a series of progesterone compounds (17-20) turned out to be highly active inhibitors of the human enzyme. The most active compound was Z-21-hydroxyiminopregna-5, 17(20)-dien-3 beta-ol (9) showing K(i) values of 44 and 3.4 nM for the human and rat enzymes, respectively, and a type II UV-difference spectrum indicating a coordinate bond between the oxime group and the heme iron. In contrast to the pregnenolones which showed no inhibition of 5 alpha-reductase isozymes 1 and 2, the progesterones 16, 17, 20, 21, and 23 showed marked inhibition, especially toward the type 2 enzyme. Compounds 17 and 20 were identified as potent dual inhibitors of both P450 17 and 5 alpha-reductase. Tested for selectivity, the most potent P450 17 inhibitors 9, 10, and 14 showed no or only marginal inhibition of P450 arom, P450 scc, and P450 TxA(2). Selected compounds were tested for inhibition of the target enzymes using whole-cell assays. Compounds 9-11 strongly inhibited P450 17 being coexpressed with NADPH-P450 reductase in E. coli cells, and 16, 20, and 23 markedly inhibited 5 alpha-reductase expressed in HEK 293 cells. Tested for in vivo activity, 9 (0.019 mmol/kg) decreased the plasma testosterone concentration in rats after 2 and 6 h by 57% and 44%.

Immunolocalization of 5alpha-reductase isozymes in acne lesions and normal skin.

Dihydrotestosterone mediates androgen-dependent diseases, such as acne, hirsutism, and androgenetic alopecia. This hormone is produced from testosterone by the 5alpha-reductase enzyme. There are 2 isozymes of 5alpha-reductase (types 1 and 2) that differ in their localization within the body and even within the skin. Activity of the type 1 isozyme predominates in sebaceous glands, where it may be involved in regulation of sebum production. Since specific inhibition of 5alpha-reductase type 1 may represent a novel therapeutic approach to acne, it is important to define the localization of these isozymes in normal sebaceous follicles and acne lesions. Skin biopsy specimens were obtained from the backs of 11 subjects: 8 with acne and 3 without acne. Sections of normal follicles, open comedones, closed comedones, and inflammatory lesions were incubated with antibodies to types 1 and 2 5alpha-reductase. In all samples, the type 1 antibody localized specifically to sebaceous glands, and the type 2 antibody localized to the companion layer of the hair follicle (the innermost layer of the outer root sheath) and granular layer of the epidermis. Localization of the type 2 isozyme was also noted within the walls of open and closed comedones and in endothelial cells from sections of inflammatory lesions. The immunolocalization of 5alpha-reductase isozymes in normal sebaceous follicles and acne follicles is similar to the pattern described in terminal hair follicles and corresponds with the findings of biochemical studies that have demonstrated predominance of type 1 activity in sebaceous glands. The function of type 2 5alpha-reductase in comedones or endothelial cells in inflammatory lesions is unknown.