Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.

Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.

Objective— Recently, mice made deficient in growth arrest–specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. Methods and Results— Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine–induced pulmonary thromoembolism and inhibited ferric chloride–induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. Conclusions— Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.

Skin disorders such as acne, seborrhea, hirsutism, and androgenic alopecia are secondary to excess local androgenic activity. Because the most potent androgen, dihydrotestosterone, is formed from testosterone by the action of 5 alpha-reductase, the inhibition of 5 alpha-reductase is a logical approach to interfere with androgenic action in the skin. In this study, we have investigated the inhibitory effect of a series of 17 beta-(N-alkyl/arylformamido)- and 17 beta-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5 alpha-androstan-3-one derivatives as 5a-reductase inhibitors following their topical application on the flank organs and ears of Golden Syrian hamsters, The parameters measured were mainly the size of the underlying sebaceous glands and 5a-reductase activity in the flank organs and ears, We found that 17 beta-(N-amylformamido)-4-methyl-4-aza-5 alpha-androstan-3-one (EM-401), 17 beta-(N-hexylformamido)-4-methyl-4-aza-5 alpha-androstan-3-one (EM-402), and 17 beta-(N-heptylformamido)-4-methyl-4-aza-5 alpha-andro-stan-3-one (EM-540) are potent inhibitors of 5a-reductase activity. EM-402 decreases the size of treated flank organs by 22%, 31%, and 32% (p < 0.01 for all) after topical application at the doses of 30, 100, and 300 mu g, respectively, twice daily for 4 wk, EM-402 also reduced the size of underlying sebaceous glands by 38%, 42%, and 59% of intact control values at the same doses. Comparable results were observed on the size of the sebaceous glands of the ears. In addition, we have observed a concentration-dependent 47%-80% (p < 0.01) and 46%-80% (p < 0.01) inhibition of 5 alpha-reductase activity in the right flank organs and ears, respectively, using topical EM-402, EM-402 had no significant effect on the same parameters in the left contralateral flank organs or ears. In addition, EM-402 had no effect on prostatic and seminal vesicle weights whereas EM-401 and EM-540 showed some systemic effects. These data illustrate that EM-402 applied topically, at the concentrations used, exerts a potent local anti-androgenic effect without any systemic action in the hamster.

Skin disorders such as acne, seborrhea, hirsutism, and androgenic alopecia are secondary to excess local androgenic activity. Because the most potent androgen, dihydrotestosterone, is formed from testosterone by the action of 5α-reductase, the inhibition of 5α-reductase is a logical approach to interfere with androgenic action in the skin. In this study, we have investigated the inhibitory effect of a series of 17β-(N-alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one derivatives as 5α-reductase inhibitors following their topical application on the flank organs and ears of Golden Syrian hamsters. The parameters measured were mainly the size of the underlying sebaceous glands and 5α-reductase activity in the flank organs and ears. We found that 17β-(N-amylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-401), 17β-(N-hexylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-402), and 17β-(N-heptylformamido)-4-methyl-4-aza-5α-andro-stan-3-one (EM-540) are potent inhibitors of 5α-reductase activity. EM-402 decreases the size of treated flank organs by 22%, 31%, and 32% (p < 0.01 for all) after topical application at the doses of 30, 100, and 300 μg, respectively, twice daily for 4 wk. EM-402 also reduced the size of underlying sebaceous glands by 38%, 42%, and 59% of intact control values at the same doses. Comparable results were observed on the size of the sebaceous glands of the ears. In addition, we have observed a concentration-dependent 47%–80% (p < 0.01) and 46%–80% (p < 0.01) inhibition of 5α-reductase activity in the right flank organs and ears, respectively, using topical EM-402. EM-402 had no significant effect on the same parameters in the left contralateral flank organs or ears. In addition, EM-402 had no effect on prostatic and seminal vesicle weights whereas EM-401 and EM-540 showed some systemic effects. These data illustrate that EM-402 applied topically, at the concentrations used, exerts a potent local anti-androgenic effect without any systemic action in the hamster. Skin disorders such as acne, seborrhea, hirsutism, and androgenic alopecia are secondary to excess local androgenic activity. Because the most potent androgen, dihydrotestosterone, is formed from testosterone by the action of 5α-reductase, the inhibition of 5α-reductase is a logical approach to interfere with androgenic action in the skin. In this study, we have investigated the inhibitory effect of a series of 17β-(N-alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one derivatives as 5α-reductase inhibitors following their topical application on the flank organs and ears of Golden Syrian hamsters. The parameters measured were mainly the size of the underlying sebaceous glands and 5α-reductase activity in the flank organs and ears. We found that 17β-(N-amylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-401), 17β-(N-hexylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-402), and 17β-(N-heptylformamido)-4-methyl-4-aza-5α-andro-stan-3-one (EM-540) are potent inhibitors of 5α-reductase activity. EM-402 decreases the size of treated flank organs by 22%, 31%, and 32% (p < 0.01 for all) after topical application at the doses of 30, 100, and 300 μg, respectively, twice daily for 4 wk. EM-402 also reduced the size of underlying sebaceous glands by 38%, 42%, and 59% of intact control values at the same doses. Comparable results were observed on the size of the sebaceous glands of the ears. In addition, we have observed a concentration-dependent 47%–80% (p < 0.01) and 46%–80% (p < 0.01) inhibition of 5α-reductase activity in the right flank organs and ears, respectively, using topical EM-402. EM-402 had no significant effect on the same parameters in the left contralateral flank organs or ears. In addition, EM-402 had no effect on prostatic and seminal vesicle weights whereas EM-401 and EM-540 showed some systemic effects. These data illustrate that EM-402 applied topically, at the concentrations used, exerts a potent local anti-androgenic effect without any systemic action in the hamster. The relationship between androgenic hormones and skin physiology has been an attractive area of research for endocrinologists and dermatologists for several decades (Pochi and Straus, 1964Pochi P.E. Straus J.S. Sebum production, casual sebum levels, titrable acidity of sebum, and urinary fractional, 17-ketosteroid excretion in males with acne.J Invest Dermatol. 1964; 43: 383-388Abstract Full Text PDF PubMed Scopus (81) Google Scholar;Cunliffe and Shuster, 1969aCunliffe W.J. Shuster S. Pathogenesis of acne.Lancet. 1969 a; i: 685-687Abstract Google Scholar). It is now well recognized that acne, hirsutism, and male pattern baldness are all androgen-related disorders (Darley et al., 1982Darley C.R. Kirby J.D. Besser G.M. Munro D.D. Edwards C.R. Rees L.H. Circulating testosterone, sex hormone binding globulin and prolactin in women with the late onset or persistent acne vulgaris.Br J Dermatol. 1982; 106: 517-522Crossref PubMed Scopus (59) Google Scholar;Barth, 1988Barth J.H. Alopecia and hirsuties. Current concepts in pathogenesis and management.Drugs. 1988; 35: 83-91Crossref PubMed Scopus (6) Google Scholar). Moreover, patients suffering from acne usually present seborrhea or an excess of serum production (Cunliffe and Shuster, 1969bCunliffe W.J. Shuster S. The rate of sebum secretion in man.Br J Dermatol. 1969 b; 81: 697-704Crossref PubMed Scopus (160) Google Scholar). In addition to excess production of androgens by the ovaries and adrenals (through DHEA), a hyperandrogenic state can also result from an increase in the 5α-reduction of testosterone in the skin (Barth, 1988Barth J.H. Alopecia and hirsuties. Current concepts in pathogenesis and management.Drugs. 1988; 35: 83-91Crossref PubMed Scopus (6) Google Scholar). In fact, testosterone, the main androgen of testicular and ovarian origins, is converted into dihydrotestosterone (DHT) in the target cell by 5α-reductase (Labrie et al., 1991Labrie C. Trudel C. Li S. Martel C. Couet J. Labrie F. Combination of an antiandrogen and a 5α-reductase inhibitor: A further step towards total androgen blockade?.Endocrinology. 1991; 129: 566-568PubMed Google Scholar[BC1]). Blockade of androgenic action can be achieved through several mechanisms, e.g., by inhibiting the conversion of testosterone to DHT with 5α-reductase inhibitors, whereas the action of both testosterone and DHT may be efficiently prevented from binding to the androgen receptor by competition with a receptor-binding compound possessing no androgenic activity, i.e., a pure anti-androgen (Voigt and Hsia, 1973Voigt W. Hsia S.L. The antiandrogen action of 4-androsten-3-one 17a-carboxyl acid and its methyl ester in hamster flank organ.Endocrinology. 1973; 92: 1216-1222Crossref PubMed Scopus (78) Google Scholar;Neumann and Steinback, 1991Neumann F. Steinback H. Androgens and antiandrogens.in: Eichler O. Farh A. Herken H. Weleh A.D. Berlin Heidelberg. Springer-Verlag, New York1991: 236Google Scholar;Plewig and Luderschmidt, 1991Plewig G. Luderschmidt C. Effect of antiandrogen on sebaceous glands and acne vulgaris. Animal experiments and studies in acne patients.in: Hammerstein J. Lachnia-Fixon U. Androgenization in Women. Excerpta Medica, Amsterdam, Oxford1991: 96-102Google Scholar). In fact, a series of steroidal and nonsteroidal compounds have been tested in animals or humans (Lutsky et al., 1975Lutsky B.N. Budak M. Koziol P. Monahan M. Neri R.O. The effects of a nonsteroid antiandrogen, Flutamide, on sebaceous gland.J Invest Dermatol. 1975; 64: 412-417Abstract Full Text PDF PubMed Scopus (32) Google Scholar;La Vecchia et al., 1984La Vecchia C. Franceschi S. Decarli A. Gallus G. Tognoni G. Risk factors for endometrial cancer at different ages.J Natl Cancer Inst. 1984; 73: 667-671PubMed Google Scholar;Luderschmidt et al., 1984Luderschmidt C. Eiermann W. Jawny J. Bidlingmaier F. Ring J. 17α-propylmesterolone (SH-434): an antiandrogenic sebosuppressive substance not influencing circulating testosterone concentrations. Experimental studies in Syrian hamsters.Nauynyn Schmiedebergs Arch Pharmacol. 1984; 328: 214-218Crossref Scopus (13) Google Scholar;Weissmann et al., 1985Weissmann A. Bowden J. Frank B.L. Horwitz S.N. Frost P. Antiandrogenic effects of topically applied spironolactone on the hamster flank organ.Arch Dermatol. 1985; 121: 57-62Crossref PubMed Scopus (37) Google Scholar;Bouton et al., 1986Bouton M.M. Lecaque D. Secchi J. Tournemine C. Effect of a new topically active antiandrogen (RU38882) on the rat sebaceous gland: comparison with cyproterone acetate.J Invest Dermatol. 1986; 86: 163-167Crossref PubMed Scopus (17) Google Scholar;Brooks et al., 1991Brooks J.R. Primka R.L. Berman C. Krupa D.A. Reynolds G.F. Rasmusson G.H. Topical anti-androgenicity of a new 4-azasteroid in the hamster.Steroids. 1991; 56: 428-433Crossref PubMed Scopus (14) Google Scholar;Lookingbill et al., 1992Lookingbill D.P. Abrams B.B. Ellis C.N. et al.Inocoterone and acne.Arch Dermatol. 1992; 128: 1197-1200Crossref PubMed Google Scholar;Cusan et al., 1993Cusan L. Dupont A. Cossette M. Labrie F. Flutamide in the treatment of female androgenic alopecia.Can J Dermatol. 1993; 5: 421-427Google Scholar,Cusan et al., 1994Cusan L. Dupont A. Gomez J.L. Tremblay R.R. Labrie F. Comparison of flutamide and spironolactone in the treatment of hirsutism: a randomized controlled trial.Fertil Steril. 1994; 61: 281-287Abstract Full Text PDF PubMed Google Scholar;Matias and Gaillard, 1995Matias J.R. Gaillard M. Local inhibition of sebaceous gland growth by topically applied (RU 58841).Ann N Y Acad Sci. 1995; 761: 56-65Crossref Scopus (7) Google Scholar;Labrie et al., 1996Labrie F. Cusan L. Dupont A. et al.In: Androgen receptor.in: Adashi E.Y. Rock J.A. Rosenwaks Z. Reproductive Endocrinology, Surgery and Technology. Lippincott-Raven Publishers, Philadelphia1996: 560-583Google Scholar). Systemically administered anti-androgens, such as 17α-methyl-B-nortestosterone and flutamide as well as the partial androgen antagonists spironolactone and cyproterone acetate, have been found to improve acne, hirsutism, and androgenic alopecia in women (Zarate et al., 1966Zarate A. Mahesh V.B. Greenblatt R.B. Effect of an antiandrogen 17α-methyl-B-nortestosterone, on acne and hirsutism.J Clin Endocrinol Metab. 1966; 26: 1394-1398Crossref PubMed Scopus (22) Google Scholar;Lookingbill et al., 1992Lookingbill D.P. Abrams B.B. Ellis C.N. et al.Inocoterone and acne.Arch Dermatol. 1992; 128: 1197-1200Crossref PubMed Google Scholar;Cusan et al., 1993Cusan L. Dupont A. Cossette M. Labrie F. Flutamide in the treatment of female androgenic alopecia.Can J Dermatol. 1993; 5: 421-427Google Scholar,Cusan et al., 1994Cusan L. Dupont A. Gomez J.L. Tremblay R.R. Labrie F. Comparison of flutamide and spironolactone in the treatment of hirsutism: a randomized controlled trial.Fertil Steril. 1994; 61: 281-287Abstract Full Text PDF PubMed Google Scholar); however, these applications are limited because of the systemic action of these anti-androgens. The pure anti-androgenic compound flutamide has been found to be more potent than the mixed androgenic/anti-androgenic compound spironolactone (Cusan et al., 1993Cusan L. Dupont A. Cossette M. Labrie F. Flutamide in the treatment of female androgenic alopecia.Can J Dermatol. 1993; 5: 421-427Google Scholar). It would be of great therapeutic value to make available drugs that have pure local anti-androgenic activity without causing systemic side-effects. Two types of human 5α-reductase, chronologically identified as type I (Andersson and Russel, 1990Andersson S. Russel D.W. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases.Proc Natl Acad Sci USA. 1990; 87: 3640-3644Crossref PubMed Scopus (482) Google Scholar;Harris et al., 1992Harris G. Azzolina B. Baginsky W. et al.Identification and selective inhibition of an isoenzyme of steroid 5α-reductase in human scalp.Proc Natl Acad Sci USA. 1992; 89: 10787-10791Crossref PubMed Scopus (126) Google Scholar) and type II (Andersson et al., 1991Andersson S. Bergman D.M. Jenkins E.P. Russel D.W. Deletion of steroid 5α-reductase 2 gene in male pseudophermaphroditism.Nature. 1991; 354: 159-161Crossref PubMed Scopus (600) Google Scholar;Labrie et al., 1992Labrie F. Sugimoto Y. Luu-the V. et al.Structure of human type II 5α-reductase.Endocrinology. 1992; 131: 1571-1573Crossref PubMed Scopus (171) Google Scholar) 5α-reductase have been isolated from human prostatic cDNA libraries, and the structure of the two isoenzymes has been elucidated. Type I 5α-reductase is predominately expressed in the skin (Andersson and Russel, 1990Andersson S. Russel D.W. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases.Proc Natl Acad Sci USA. 1990; 87: 3640-3644Crossref PubMed Scopus (482) Google Scholar;Harris et al., 1992Harris G. Azzolina B. Baginsky W. et al.Identification and selective inhibition of an isoenzyme of steroid 5α-reductase in human scalp.Proc Natl Acad Sci USA. 1992; 89: 10787-10791Crossref PubMed Scopus (126) Google Scholar;Luu-the et al., 1994Luu-the V. Sugimoto Y. Puy L. Labrie Y. Lopez I. Singh M. Labrie F. Characterization, expression and immunohistochemical localization of 5α-reductase in human skin.J Invest Dermatol. 1994; 102: 221-226Abstract Full Text PDF PubMed Google Scholar), whereas the type II enzyme is responsible for male pseudohermaphroditism when defect. We have previously reported that a series of 17β-(N-alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one derivatives strongly inhibit human type I 5α-reductase activity and show low potency on human type II 5α-reductase in transfected cells in vitro (Li et al., 1995Li X. Singh S.M. Labrie F. Synthesis and in vitro activity of 17β-(N-alkyl/arolformamido) – and 17β-[(N-alkyl/aryl) alkyl/arylamido]-4-methyl-4-aza-3-oxo-5α-androstan-3-ones as inhibitors of human 5α-reductases and antagonists of the androgen receptor.J Med Chem. 1995; 38: 1158-1173Crossref Scopus (23) Google Scholar). Because the androgen sensitivity of the Syrian hamster flank organs and ears have made this animal a useful model for the study of such compounds (Hamilton and Montagna, 1950Hamilton J.B. Montagna W. The sebaceous glands of the hamster. Morphological effects of androgens on integumentary structure.Am J Anat. 1950; 86: 191-233Crossref PubMed Scopus (70) Google Scholar;Plewig and Luderschmidt, 1982Plewig G. Luderschmidt C. Hamster ear model for sebaceous glands.J Invest Dermatol. 1982; 68: 171-176Crossref Scopus (72) Google Scholar;Vermorken et al., 1982Vermorken A.J.M. Goos C.M.A.A. Wirtz P. Evaluation of the hamster flank organ test for the screening of antiandrogens.Br J Dermatol. 1982; 106: 99-101Crossref PubMed Scopus (30) Google Scholar;Matias and Gaillard, 1995Matias J.R. Gaillard M. Local inhibition of sebaceous gland growth by topically applied (RU 58841).Ann N Y Acad Sci. 1995; 761: 56-65Crossref Scopus (7) Google Scholar), we have evaluated the anti-androgenic activity of these compounds on Syrian hamster flank organs and ears after a 4 wk topical administration. Male Syrian hamsters ≈8–10 wk old and weighing 110–120 g were purchased from Charles-River Laboratories (St. Constant, Québec, Canada) and housed 4–5 per cage in a light (14 h light per d, lights on at 07:15) and temperature (21 ± 3°C) controlled environment. The animals were housed in plastic boxes on sawdust and received hamster chow (Agway Country Food, Syracuse, NY) and tap water ad libitum. At the beginning of the experiment, the ear was marked for identification purposes and the area of the flank organ was shaved with electric clippers. The appropriate doses of the indicated compounds were dissolved in ethanol-propylene glycol (50:50, vol/vol) and administered in 10 μl on the right side of the hamster flank organs as well as the ears. Meanwhile, the left side of the flank organs and ears of the animals of the control and treated groups received the vehicle only. The three experiments were performed as follows: Experiment 1Intact hamsters (four per group) were treated with EM-423, 347, 401, 402, 422, 435, 336, 337, 436, 540, 541 at a dose of 50 μg, twice daily, on the right flank organ. Experiment 2Intact hamsters (four per group) were treated with EM-424, 486, 497, 494, 493, 498, 503, 580, 568, 606, 567, 682 at a dose of 100 μg, twice daily, on the right flank organ. Experiment 3Intact hamsters (12 per group) were treated with EM-401, 402, and 540 at doses of 30, 100, and 300 μg, twice daily, on the right flank organ and ear. The 5α-reductase inhibitors were administered twice daily for 28 consecutive days. The length and width of the darkly pigmented oval spot outlining each flank organ were carefully measured with Vernier calipers (Fisher Scientific, Pittsburgh, PA). Flank organ area was then calculated according to the following formula: area of ellipse = π(L/2 × W/2) = 3.14×(L × W)/4. The animals were killed by decapitation on the morning following the last dose. The flank organs and ears were then removed quickly and fixed for histologic examination or frozen in dry ice, and stored at –80°C for enzymatic assays. The ventral prostate as well as the seminal vesicles were removed, dissected, freed from fat and connective tissue, and rapidly weighed. The flank organs and ears were fixed in 4% paraformaldehyde for 48 h and then rinsed in 15% sucrose phosphate buffer for 24 h at 4°C. The flank organs were then cut in the middle in the rostral to caudal direction and the ears were cut between the first and second cartilage ridges. The tissues were then embedded in tissue-tek (Miles, Diagnostic Division, Elkhart, IN) and cut with a cryostat in 8 μm thick sections before staining with hematoxylin and eosin. The surface areas of the sebaceous glands of flank organs were estimated with a computer-assisted Image-Pro Plus program (Media Cybernetics, Silerspring, MD), and the surface areas of the sebaceous glands of the inner surface of the ears were estimated directly under optical microscopy using a grid. The surface area was counted as square units occupied by sebaceous glands in each section area. Six consecutive sections of each sample were analyzed for both flank organ and ear. The total number of arbitrary units was counted and the mean of six slides from each sample was calculated. Only the glands of the inner surface of the ear were measured. The results were calculated in units and expressed as a percentage of the control group that was taken as 100%. The enzymatic assay was performed as described previously (Martel et al., 1994Martel C. Melner M.H. Gagné D. Simard J. Labrie F. Widespread tissue distribution of steroid sulfatase, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), 17β-HSD 5α-reductase and aromatase activities in the rhesus monkey.Mol Cell Endocrinol. 1994; 104: 103-111Crossref PubMed Scopus (184) Google Scholar). In brief, 100 μl aliquots of the 1000 ×g supernatant of hamster flank organs and ears were incubated for 3 h at 37°C in a total volume of 0.5 ml phosphate buffer (12.5 mM KH2PO4, 1 mM ethylenediamine tetraacetic acid, pH 7.5) containing 0.5 μM [4–14C]-testosterone (S.A., 51.4 mCi per mmol) and the cofactor NADPH. Labeled radioactivity was purchased from New England Nuclear/Dupont (Markham, Canada) and purified by thin-layer chromatography before use. The enzymatic reaction was stopped by chilling the incubation mixture in an ice-water slurry and adding 3 ml of diethyl ether. The components were then mixed and frozen in a dry ice–ethanol bath. The organic phase was kept while the remaining frozen aqueous fraction was re-extracted once with ether. The organic phases were then pooled and evaporated to dryness under a nitrogen stream. All components were then separated on thin-layer chromatography (60 F254 silica gel, Merck, Darmstadt, Germany) using toluene:acetone (4:1, vol/vol) as solvent before autoradiography of the plates for 48 h. The metabolites revealed by autoradiography were identified by comparison with standard labeled steroids. The thin-layer chromatography areas corresponding to testosterone, DHT, and the DHT metabolites androstane-3α,17β-diol and androstane-3β,17β-diol were scraped and transferred into vials containing 0.5 ml ethanol and 10 ml scintillation liquid. The radioactivity was then measured in a liquid scintillation counter. Results are expressed as means ± SEM in pM product formed per mg protein per min. Statistical significance was measured according to the multiple-range test of Duncan-Kramer (Kramer, 1956Kramer C.Y. Extension of multiple range tests to group means with unique numbers of replications.Biometrics. 1956; 12: 307-310Crossref Google Scholar). Data are expressed as means ± SEM. Novel 17β-(N-alkyl/aryl formamido)-4-methyl-4-aza-5α-androstan-3-ones show topical anti-androgenic activityA series of novel 17β-(N-alkyl/arylformamido)-4-methyl-4-aza-5α-androstan-3-ones (Figure 1) were tested by application on hamster flank organs. The compounds were administered topically on the right flank organs of Golden Syrian hamsters at a dose of 50 μg, twice daily, for 4 wk, whereas the control animals and the left side of the animals of the treated groups received the vehicle only. As can be seen in Table 1, the surface area of the right side flank organs (treated side) of hamsters treated with EM-347, EM-402, EM-540, EM-337, EM-436, and EM-435 decreased by 27% (p < 0.01), 26% (p < 0.01), 24% (p < 0.01), 17% (p < 0.05), 21% (p < 0.05), and 25% (p < 0.01), respectively, whereas the surface area of the left side flank organs (the side treated with vehicle only) was unaffected. On the other hand, compounds EM-401 and EM-541 demonstrated systemic activity because they reduced the surface area of both side flank organs by 24% (p < 0.01), 26% (p < 0.01), 18% (p < 0.01), and 25% (p < 0.01), respectively. Treatment with EM-423, EM-422, and EM-336 did not affect flank organ size. It can be seen in the same table that the weights of the ventral prostates and seminal vesicles were not significantly reduced by treatment with any of the compounds used.Table IInhibitory effects of 17β-(N-alkylformanido)-4-methyl-4-aza-5α-androstan-3-one derivatives on the size of flank organs as well as ventral prostate and seminal vesicle weight. Compounds were applied on the right flank organaData are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls.EM(50 mg, BID)Ventral prostate weight (mg)Seminal vesicle weight (mg)Left sideRight sideControl33.5 ± 2.133.4 ± 1.353.9 ± 4.1228.4 ± 6.442334.9 ± 3.134.8 ± 1.355.3 ± 4.0242.7 ± 6.934732.0 ± 1.324.6 ± 1.0**55.0 ± 3.2208.0 ± 12.140125.3 ± 1.8**24.6 ± 2.0**54.1 ± 1.6249.5 ± 17.040232.1 ± 0.924.6 ± 1.2**50.0 ± 0.9226.5 ± 25.554032.9 ± 0.825.1 ± 0.7**50.7 ± 3.0248.9 ± 6.454127.5 ± 1.4*25.0 ± 0.5**55.5 ± 1.0256.0 ± 3.642230.4 ± 2.228.2 ± 1.858.2 ± 2.8227.3 ± 22.343529.3 ± 0.925.2 ± 1.3**59.0 ± 6.0223.9 ± 20.733632.3 ± 0.633.2 ± 1.149.3 ± 4.5212.1 ± 10.233732.4 ± 1.227.7 ± 0.8*55.4 ± 2.1221.7 ± 23.843636.1 ± 1.826.3 ± 1.7*58.4 ± 3.1214.3 ± 23.0a Data are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls. Open table in a new tab A series of 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-ones (Figure 1) were also tested on hamster flank organs. As illustrated in Table 2, the topical administration of EM-486, EM-568, EM-606, and EM-567, at a dose of 100 μg, twice daily, for 4 wk, caused respective 21% (p < 0.05), 30% (p < 0.01), 29% (p < 0.01), and 28% (p < 0.05) reductions in the left flank organ size and 26%, 25%, 29%, and 29% (p < 0.01 for all) reductions in the right side flank organ surface area compared with the intact control group. Ventral prostate weights were decreased by 18% (p < 0.05) and 27% (p < 0.01) following treatment with EM-568 and EM-606, respectively, whereas EM-486 did not affect this parameter. Moreover, treatment with EM-568 and EM-606, at the same dose, caused 23% and 33% (p < 0.01 for both) reductions in seminal vesicle weight, although EM-468 and EM-567 had no significant effect on this parameter. On the other hand, although EM-682 did not significantly affect flank organ surface area, ventral prostate and seminal vesicle weights were reduced by 23% and 22% (p < 0.01), respectively, with topical treatment at the twice daily dose of 100 μg. As can be seen in Table 2, treatment with EM-424, EM-497, EM-494, EM-498, EM-503, EM-493, and EM-580 did not affect the surface area of either flank organs or prostate and seminal vesicle weight.Table IIInhibitory effects of 17β-[N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one series on the size of flank organ, ventral prostatic, and seminal vesicle weights. Compounds were applied on the right flank organaData are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls.EM(100 μg, BID)Ventral prostate weight (mg)Seminal vesicle weight (mg)Left sideRight sideControl33.2 ± 2.033.6 ± 1.655.4 ± 3.1236.3 ± 13.242431.9 ± 1.332.8 ± 1.656.5 ± 4.4265.4 ± 15.848626.3 ± 0.6*25.0 ± 1.3**59.3 ± 7.4267.1 ± 16.649730.4 ± 1.230.4 ± 0.859.1 ± 2.8249.3 ± 5.349431.1 ± 1.130.7 ± 0.953.8 ± 3.3240.1 ± 5.349331.7 ± 2.127.3 ± 1.457.8 ± 2.0238.3 ± 6.349830.4 ± 1.632.3 ± 2.153.1 ± 3.5248.8 ± 8.350331.9 ± 1.230.0 ± 1.055.1 ± 1.6277.1 ± 11.258030.2 ± 1.132.1 ± 1.253.7 ± 3.2224.2 ± 4.856823.2 ± 0.7**25.3 ± 1.5**45.7 ± 3.5*182.7 ± 7.5**60623.5 ± 2.3**23.8 ± 2.2**40.3 ± 3.4**157.7±11.6**56724.0 ± 1.6**24.0 ± 1.4**51.3 ± 1.4211.8 ± 6.968228.1 ± 1.831.1 ± 1.442.9 ± 2.0**183.3 ± 8.2**a Data are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls. Open table in a new tab In order to obtain more precise information on the activity of the compounds potentially suitable for topical use and devoid of systemic activity, we measured the effects of EM-401, EM-402, and EM-540 on the size of the flank organs and the size of the sebaceous glands of flank organs and ears, 5α-reductase activity of flank organs and ears, as well as the weight of the prostate and seminal vesicle. The compounds were applied topically on the right flank organ and ear at doses of 30, 100, or 300 μg, twice daily. As illustrated in Figure 2, the surface areas of the flank organs of intact control animals measured 32.7 ± 1.0 and 33.4 ± 1.22 mm2 for the left and right sides, respectively. In hamsters treated with EM-401 at doses of 30, 100, and 300 μg, twice daily, for 4 wk, left and right flank organs measured 28.1 ± 1.1 and 29.2 ± 1.7 mm2, 26.0 ± 1.4 and 25.0 ± 1.5 mm2, and 25.3 ± 2.0 and 21.9 ± 0.7 mm2. On the other hand, treatment with increasing doses of EM-402 decreased the size of the right flank organ to the values of 26.0 ± 1.2, 22.8 ± 1.3, and 22.6 ± 1.9 mm2, whereas the size of left flank organs remained within the range of intact controls. Similar results were observed in the animals treated with EM-540 where right flank organ size decreased to 26.7 ± 1.2, 25.3 ± 1.6, and 20.7 ± 1.0 mm2 following doses of 30, 100, and 300 μg, respectively, whereas the left flank organ did not change significantly. Figure 3 illustrates the changes in the size of the sebaceous glands underlying the hamster flank organs that may be considered as a more clinically relevant parameter than the change in size of the flank organ itself. Treatment with 30, 100, and 300 μg EM-401 reduced the size of the left flank sebaceous glands to 89.3% (not significant), 66.7% (p < 0.01), and 63.4% (p < 0.01) of control and that of the right flank sebaceous glands to 94.4% (not significant), 42.0% (p < 0.01), and 24.0% (p < 0.01) of control, respectively. In contrast, treatment with EM-402 and EM-540 reduced the size of the flank organ sebaceous glands on the treated side only, whereas contralateral glands were unaffected. In fact, EM-402 at the dose of 30 μg decreased right sebaceous gland size to 58% (p < 0.01) of control, whereas the doses of 100 and 300 μg twice daily, led to values of 61.6% and 40.8% (p < 0.01) of control, respectively. On the other hand, the inhibitory effect of EM-540 decreased right sebaceous gland size to 53% (p < 0.01) for the smallest dose used (30 μg), whereas the larger doses of 100 and 300 μg resulted in further decreases to 56% and 45% (p < 0.01) of control, respectively. The inhibitory effects of EM-401, EM-402, and EM-540 were also examined on the size of the sebaceous glands of ears, because the inner surface of the hamster ear contains large sebaceous glands that are highly sensitive to androgens. As can be seen in Figure 4, the effects of the three compounds were comparable with those obtained on the size of the sebaceous glands of the flank organs. In fact, topical treatment with EM-401 at the dose of 30 μg decreased the size of sebaceous glands of the right ear to 57.6 ± 6.9% (p < 0.01) of control, whereas the left side was unaffected. Following twice daily treatment with 100 and 300 μg of EM-401, 91.4 ± 4.3% and 73.1 ± 6.2% (p < 0.01) of control values were measured on the left side and 36.5 ± 3.1% and 31.9 ± 3.1% (p < 0.01) of control values were measured on the right side, respectively. On the other hand, EM-402 decreased right ear sebaceous gland size to 48.0 ± 3.6%, 38.9 ± 4.8%, and 48.8 ± 5.1% (p < 0.01) of control intact values and EM-540 decreased gland size to 73.8 ± 5.9%, 54.1 ± 4.7%, and 43.0 ± 2.2% at the doses of 30, 100 and 300 μg, respectively. Neither of the two compounds affected gland size in untreated ears. As we reported previously, 17β-(N-Alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-ones are potent inhibitors of transfected human type I 5α-

OBJECTIVES:Because the large increase in luteinizing hormone secretion induced by flutamide in the intact rat is not found in men, we have used castrated rats and mice supplemented with androstenedione (4-dione) instead of intact animals to measure the activity of the pure antiandrogens flutamide and Casodex.METHODS:We first compared the effect of different schedules of administration of various doses of the two antiandrogens on prostate and seminal vesicle weights in the castrated rat and mice models.RESULTS:For both flutamide and Casodex, no consistent difference was found between the effects of once daily and thrice daily oral dosing in the rat. It was observed, however, that flutamide, especially at the high and therapeutically more effective doses, is about three times more potent than Casodex under both schedules of dosing. When flutamide was administered subcutaneously three times a day, twice a day, once a day, or once every second day in rats and mice, no difference was observed in the degree of inhibition achieved on prostate and seminal vesicle weights.CONCLUSIONS:The present data show that Casodex is about three times less potent than flutamide on the well-recognized parameters of androgen responsiveness in the rat, namely prostate and seminal vesicle weights. Another finding is that once daily dosing with flutamide exhibits an effectiveness comparable to thrice daily dosing; such data may have potential significance in facilitating compliance by administration of flutamide once daily instead of the current thrice daily schedule in men. Moreover, these data, if obtained in a reliable in vivo model, should be helpful in determining the choice of an appropriate dose of Casodex for the treatment of prostate cancer.

A quantitative in situ hybridization study was carried out to determine the precise localization and androgen regulation of the flank organ regulated (FAR-17A) mRNA expression in the different cellular components of the hamster flank organs. Although FAR-17A mRNA was highly expressed in the epithelial cells of the sebaceous glands, it was also found in the outer root sheath of the hair follicles and in melanocytes. The changes in FAR-17A mRNA levels, in the size of the flank organ and sebaceous gland areas as well as in the weight of the seminal vesicles and prostate, were compared following castration and after 5alpha-dihydrotestosterone treatment. FAR-17A mRNA levels were already significantly decreased 1 d after castration, in parallel with a concomitant decrease in the number of labeled cells with the FAR-17A probe. A maximal decrease was found 7 d after castration. The other parameters were significantly reduced later. After 7 d of treatment with dihydrotestosterone, all values returned to those found in intact animals. Similar stimulatory effects on these parameters were observed after treatment with the adrenal sex steroid precursor dehydroepiandrosterone. These data show that all of the components of the flank organs (sebaceous glands, hair follicles, and melanocytes) express the flank organ regulated (17A) type gene (FAR-17A) gene and that its expression is stimulated by treatment with either dihydrotestosterone or dehydroepiandrosterone. Moreover, FAR-17A mRNA levels respond to androgen stimulation more rapidly than the standard morphologic parameters, revealing that the FAR-17A gene could be a more sensitive and cell specific marker to study the mechanisms of androgen action in the skin.

Cancer of the prostate is the most frequent cancer and the second leading cause of cancer death in men in North America. The growth of Shionogi carcinoma-115 (SC-115) cells is highly sensitive to androgens, and this cell line is a well known experimental model of prostate cancer. The transplantable Shionogi carcinoma tumor was used to assess the influence of tumor size on the response to flutamide treatment. Two weeks after subcutaneous inoculation of tumor fragments in Shionogi mice, six groups of animals bearing SC-115 tumors ranging from 0.1 to 1.8 cm in diameter were treated with flutamide (1 mg, twice daily). The castrated mice received an androstenedione (delta4-dione) implant to mimic the human situation, where the adrenals produce precursor steroids which are transformed into androgens in peripheral intracrine tissues. After 16 days, treatment with flutamide inhibited tumor growth by 32 to 57% in the four groups of mice having tumors ranging from 0.1 to 1.0 cm in diameter at day 0, whereas no significant inhibitory effect was observed in larger tumors. The same treatment, however, caused potent inhibitory effects on other androgen-sensitive parameters, namely prostatic and seminal vesicle weight and kidney ornithine decarboxylase (ODC) activity, the effect on these parameters being similar in all groups of animals, irrespective of tumor size. Furthermore, when those larger tumors unresponsive to antiandrogenic treatment were cut into small fragments and inoculated into new groups of mice, the same treatment with flutamide efficiently inhibited tumor growth, treatment being started at tumor sizes of 0.1 to 0.3 cm in diameter. The present data clearly demonstrate that small tumors are highly sensitive to androgen deprivation, while loss of response develops with increasing tumor size, thus indicating that, for optimal efficacy, androgen blockade should be given at the early stages of prostate cancer.

Since evidence of 5 alpha-reductase activity in rabbit liver homogenate was discovered in 1954, the presence of this enzyme has been demonstrated in many other organs and tissues of mammalian species. 5 alpha-Reductase selectively transforms a 4-ene-3-oxosteroid (e.g., testosterone) irreversibly to the corresponding 5 alpha-3-oxosteroid (e.g., 5 alpha-dihydrotestosterone) in the presence of NADPH as an essential coenzyme at an optimal pH. However, excessive production of 5 alpha-dihydrotestosterone is the major cause of many androgen-related disorders, such as prostate cancer, benign prostatic hyperplasia, acne, female hirsutism, and male pattern baldness; therefore, inhibition of androgenic action by 5 alpha-reductase inhibitors is a logical treatment. During the past two decades, research has focused on understanding the biological functions and effects of 5 alpha-reductase and its 5 alpha-reduced metabolites: purification of the enzyme, substrates, and metabolites; characterization of their physical, chemical, and biochemical properties; analysis of the amino acid sequence of the enzyme; synthesis of various classes of molecules as potential inhibitors; and examination of the biological activity of the inhibitors in vitro and/or in vivo. This review summarizes the biochemical studies on this enzyme, suggests the mechanisms of action of the enzyme or inhibitors, and discusses the chemistry necessary for the preparation, structure-activity relationships, and in vitro and/or in vivo data obtained from the evaluation of nonsteroidal and steroidal compounds that have been tested as inhibitors of 5 alpha-reductase. In particular, IC50 and K-i values for relevant compounds will be compared according to molecular class. This review could function as a comprehensive working reference of what research has been accomplished so far and what problems remain to be solved in the future for those engaged in this interesting field.

The hamster flank organ is a widely used model of the control of sebaceous gland activity by androgens and anti-androgens. Finasteride, a 5 alpha-reductase inhibitor, was administered locally on the surface of the right flank organ and right ear twice daily for 4 weeks. The treatment caused similar 12% to 30% reductions in the size of the sebaceous glands in both flank organs. Moreover, relative mRNA levels of the androgen-regulated FAR-17a gene measured by in situ hybridization as well as [3H]-thymidine incorporation and 5 alpha-reductase activity were similarly decreased in the two flank organs after topical application. The pure anti-androgen flutamide, at the same doses, exerted a more potent effect on all the same parameters, and the effect was also comparable on both the treated and untreated sides of flank organs. Finasteride and flutamide significantly decreased ventral and dorsal prostatic weights after topical application. The present data show that the topical administration of finasteride, in analogy with flutamide, causes local inhibition of sebaceous gland growth in both the costovertebral organs and ears. However, as demonstrated by the similar inhibitory effect in the contralateral untreated side and the reduced weight of the dorsal and ventral lobes of the prostate and seminal vesicles, finasteride and flutamide both exert significant systemic effects.